Human monoclonal antibody neutralizing vascular endothelial growth factor receptor and use thereof

ABSTRACT

The present invention relates to human monoclonal antibodies neutralizing vascular endothelial growth factor receptor and the use thereof. More specifically, relates to human ScFv molecules neutralizing vascular endothelial growth factor receptor, and a composition for inhibiting angiogenesis and a composition for treating cancer, which contain the human ScFv molecules. The disclosed monoclonal antibody neutralizing vascular endothelial growth factor receptor shows excellent neutralizing ability in living cells, compared to that of a commercially available antibody against vascular endothelial growth factor receptor, and shows the ability to neutralize vascular endothelial growth factor receptor not only in humans, but also in mice and rats. Thus, the monoclonal antibody will be useful in anticancer studies and will be highly effective in cancer treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/664,226, filed on Nov. 19, 2010 (currently pending), thedisclosure of which is herein incorporated by reference in its entirety.The U.S. patent application Ser. No. 12/664,226 is a national entry ofInternational Application No. PCT/KR2007/003077, filed on Jun. 26, 2007,which claims priority to Korean Application No. 10-2007-0057719 filed onJun. 13, 2007, the entire contents of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to human monoclonal antibodiesneutralizing vascular endothelial growth factor receptor and the usethereof, and more particularly to human ScFv molecules neutralizingvascular endothelial growth factor receptor, and a composition forinhibiting angiogenesis and a composition for treating cancer, whichcontain the human ScFv molecules.

BACKGROUND ART

Angiogenesis means the formation of new blood vessels from pre-existingvessels by the growth, differentiation and migration of endothelialcells and does not occur in healthy adults, except for some specialoccasions, including wound healing, menstruation, etc. However, theexcessive formation of new blood vessels in diseases, such as tumorgrowth and metastasis, age-related macular degeneration, rheumatoidarthritis, diabetic retinopathy, psoriasis and chronic inflammation, hasbeen reported (Cameliet and Jain, Nature, 407:249, 2000). For thisreason, many efforts to treat diseases, particularly tumors, usingangiogenesis inhibitors, have been made.

Factors involved in angiogenesis include vascular endothelial growthfactor (VEGF), epithelial growth factor (EGF), platelet-derived growthfactor (PDGF), transforming growth factor-b (TGFb), fibroblast growthfactor (FGF), etc. Among them, the vascular endothelial growth factor isan endothelial cell-specific factor which is involved directly in thegrowth, differentiation and migration of endothelial cells, and thereare four different isoforms (VEGF165, VEGF121, VEGF189 and VEGF206).Among the four isoforms, VEGF165 is the most abundant isoform in allhuman tissues except placenta (Tisher et al., J. Biol. Chem., 266:11947,1991).

Vascular endothelial growth factor (VEGF) regulates new blood vesselformation resulting from the differentiation of endothelial precursors(angioblasts) in situ, is expressed in embryonic tissues (Breier et al.,Development (Camb), 114:521, 1992), macrophages, and proliferatingepithelial keratinocytes during wound healing (Brown et al., J. Exp.Med., 176:1375, 1992), and may be responsible for tissue edemaassociated with inflammation (Ferrara et al., Endocr. Rev., 13:18,1992). In situ hybridization studies have demonstrated high VEGFexpression in a number of human tumor lines including glioblastomamultiforme, hemangioblastoma, central nervous system neoplasms andAIDS-associated Kaposi's sarcoma (Plate et al., Nature, 359:845, 1992;Plate et al., Cancer Res., 53: 5822, 1993; Berkman et al., J. Clin.Invest., 91:153, 1993; Nakamura et al., AIDS Weekly, 13(1), 1992). Highlevels of VEGF were also observed in hypoxia induced angiogenesis(Shweiki et al., Nature, 359:843, 1992).

The biological function of VEGF is mediated through its high affinityVEGF receptors which are selectively expressed in endothelial cellsduring embryogenesis (Millauer et al., Cell, 72:835, 1993) and duringtumor formation. VEGF receptors (VEGFR) typically are class IIIreceptor-type tyrosine kinases characterized by having several,typically 5 or 7, immunoglobulin-like loops in their amino-terminalextracellular ligand-binding domain of a receptor (Kaipainen et al., J.Exp. Med., 178:2027, 1993). The other two regions include atransmembrane region and a carboxy-terminal intracellular catalyticdomain interrupted by an insertion of hydrophilic interkinase sequencesof variable lengths, called the kinase insert domain (Terman et al.,Oncogene, 6:1677, 1991). VEGF receptors include fms-like tyrosine kinasereceptor (Flt-1), or VEGFR-1 (Shibuya et al., Oncogene, 5:519, 1990; WO92/14248; Terman et al., Oncogene, 6:1677, 1991), kinase insertdomain-containing receptor/fetal liver kinase (KDR/Flk-1), or VEGFR-2(Matthews et al., PNAS, 88:9026, 1991), although other receptors such asneuropilin-1 and neuropilin-2 can also bind VEGF. Another tyrosinekinase receptor, VEGFR-3 (Flt-4), binds the VEGF homologues VEGF-C andVEGF-D and is important in the development of lymphatic vessels.

High levels of Flk-1 are expressed by endothelial cells that infiltrategliomas (Plate et al., Nature, 359:845, 1992). Flk-1 levels arespecifically upregulated by VEGF produced by human glioblastomas (Plateet al., Cancer Res., 53:5822, 1993).

The finding of high levels of Flk-1 expression in glioblastomaassociated endothelial cells (GAEC) indicates that receptor activity isprobably induced during tumor formation since Flk-1 transcripts arebarely detectable in normal brain endothelial cells. This upregulationis confined to the vascular endothelial cells in close proximity to thetumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonalantibodies (mAbs) resulted in inhibition of the growth of human tumorxenografts in nude mice (Kim, K. et al., Nature, 362:841-844, 1993),indicating a direct role for VEGF in tumor-related angiogenesis.

Although VEGF ligands are upregulated in tumor cells, and the receptorsthereof are upregulated in tumor infiltrated vascular endothelial cells,the expression levels of VEGF ligands and the receptors thereof are lowin normal cells that are not associated with angiogenesis. Therefore,such normal cells would block the interaction between VEGF and thereceptors thereof to inhibit angiogenesis, thus inhibiting tumor growth.

High levels of VEGFR-2 are expressed by endothelial cells thatinfiltrate gliomas, and are specifically upregulated by VEGF produced byhuman glioblastomas (Plate et al., Nature, 359:845, 1992; Plate et al.,Cancer Res., 53:5822, 1993). The finding of high levels of VEGR-2expression in glioblastoma associated endothelial cells (GAEC) suggeststhat receptor activity is induced during tumor formation, since VEGFR-2transcripts are barely detectable in normal brain endothelial cells.

Therefore, studies focused on inhibiting the activity of VEGF, which isexpressed in tumor growth sites, to inhibit angiogenesis so as toinhibit tumor growth, are being actively conducted. Typically, methodsof inhibiting VEGF receptors on the membrane of cancer cells to preventVEGF from entering cells have been developed. Examples of cell linesproducing VEGFR antibodies include a hybridoma cell line producing ratanti-mouse VEGFR-2 monoclonal antibody (DC101; ATCC HB 11534), ahybridoma cell line (M25, 18A1; ATCC HB 12152) producing mouseanti-mouse VEGFR-2 monoclonal antibody mAb 25, and a hybridoma cell lineproducing mouse anti-mouse VEGFR-2 monoclonal antibody mAb 73 [(M73,24;ATCC HB 12153), KM1730(FERM BP-5697; WO 98/22616; WO 99/59636), KM1731(FERM BP-5718), KM1732 (FERM BP-5698), KM1748 (FERM BP-5699), KM1750(FERM BP-5700)].

There has been a continuous development of humanized antibodies againstVEGF receptors. These humanized antibodies against VEGF receptors,developed to date, showed high competition with VEGF in vitro, but hadproblems in that their ability to neutralize VEGF receptors in cells isreduced and in that the antibodies do not show cross-reactivity in miceor rats, such that animal tests cannot be carried out.

Accordingly, the present inventors have constructed a library ofnon-immunized fully human antibodies, screened single chain variablefragment (ScFv) antibodies against VEGF receptor (KDR), and found thatthe antibodies exhibit an excellent KDR-neutralizing effect not only invitro, but also in cells and in vivo, and show cross-reactivity even inmice and rats, thereby completing the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a fully human singlechain variable fragment (ScFv) antibodies 6A6-ScFv and 6A6-IgG, whichhave an excellent ability to neutralize VEGF receptor in cells and invivo.

Another object of the present invention is to provide a fully humansingle chain variable fragment (ScFv), which is a light chain variant of6A6, which shows a more excellent ability to neutralize VEGF receptorcompared to that of 6A6-ScFv.

Still another object of the present invention is to provide acomposition for inhibiting angiogenesis, which contains a fully humanScFv or IgG having the ability to neutralize VEGF receptor.

Yet another object of the present invention is to provide a compositionfor treating cancer, which contains a fully human ScFv or IgG having theability to neutralize VEGF receptor.

To achieve the above objects, in one aspect, the present inventionprovides a single chain variable fragment (ScFv) molecule, whichcontains a light chain variable region represented by an amino acidsequence of any one of SEQ ID NOS: 1 to 19 and functions to neutralizevascular endothelial growth factor receptor. In the present invention,the ScFv (single chain variable fragment) molecule and a constructthereof preferably have a heavy chain variable region represented by anamino acid sequence of SEQ ID NO: 20.

In another aspect, the present invention provides a DNA encoding saidScFv (single chain variable fragment) molecule, a vector containing saidDNA, and recombinant cells transformed with said vector. In the presentinvention, the cells are preferably bacterial or animal cells.

In still another aspect, the present invention provides a compositionfor inhibiting angiogenesis, which contains said ScFv molecule, and acomposition for treating cancer, which contains said ScFv molecule.

In still another aspect, the present invention provides an IgG, whichcontains a light chain variable region represented by an amino acidsequence of any one of SEQ ID NOS: 1 to 19 and functions to neutralizevascular endothelial growth factor receptor. In the present invention,said IgG preferably has a heavy chain variable region represented by anamino acid sequence of SEQ ID NO: 20.

In yet another aspect, the present invention provides a composition forinhibiting angiogenesis, which contains said IgG, and a composition fortreating cancer, which contains said IgG.

Other features and aspects of the present invention will be apparentfrom the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the amino acid sequence and function of a gene insertedinto a pcDNA3-KDR D123tFcm vector.

FIG. 2 shows a schematic diagram of KDR(ECD1-2) and KDR(ECD2-3)-Fc forepitope mapping according to the present invention.

FIG. 3 shows the results of SDS-PAGE of KDR(ECD1-3)-Fc purified in thepresent invention.

FIG. 4 shows the results of VEGF competition assays for anti-KDR phageand anti-KDR-SvFc according to the present invention.

FIG. 5 shows the nucleic acid sequence, amino acid sequence and CDRsequence of 6A6 ScFv phage according to the present invention.

FIG. 6 shows SDS-PAGE results for purified 6A6 ScFv.

FIG. 7 shows the results of VEGF competition assays using anti-KDR-ScFv.

FIG. 8 shows the results of epitope mapping of anti-KDR-ScFv accordingto the present invention.

FIG. 9 shows a cleavage map of pIGHD-6A6Hvy that is a vector containingthe invariable region and heavy chain region of 6A6 IgG.

FIG. 10 shows a cleavage map of pIgGLD-6A6Lgt that is a vectorcontaining the constant region and light chain region of IgG.

FIG. 11 shows the results of SDS-PAGE of purified 6A6 IgG.

FIG. 12 shows the results of VEGF competition assays using anti-KDR 6A6IgG.

FIG. 13 shows the results of competition assays of anti-KDR 6A6 IgG withVEGF families.

FIG. 14 shows the results of FACS analysis for the binding affinity ofthe inventive anti-KDR IgG antibody to HUVEC cells.

FIG. 15 shows FACS assay results for the competition of the inventiveanti-KDR IgG antibody with VEGF165 in HUVEC cells.

FIG. 16 shows the results of Western blot analysis for the expression ofKDR in K562 cells (ATCC CCL-243).

FIG. 17 shows the binding affinity of the inventive anti-KDR IgGantibody to the K562 cell line.

FIG. 18 shows FACS assay results for the competition of the inventiveanti-KDR IgG antibody with VEGF in the K562 cell line.

FIG. 19 shows FACS analysis results for the binding affinity of ananti-KDR antibody to a Gleevec-resistant cell line.

FIG. 20 shows analysis results for the cell proliferation inhibition ofthe anti-KDR-IgG according to the present invention.

FIG. 21 shows the results of Western blot analysis for the ability ofthe inventive anti-KDR antibody to inhibit KDR phosphorylation and ERKphosphorylation, which is induced by VEGF.

FIG. 22 shows the ability of an IgG-type KDR antibody to inhibit themigration of endothelial cells by VEGF.

FIG. 23 shows that an IgG-type KDR antibody inhibits endothelial celltube formation induced by VEGF.

FIG. 24 shows the inhibition of VEGF-KDR internalization through thebinding between the IgG-type KDR antibody and cell surface KDR.

FIG. 25 shows the inhibitory effect of the IgG-type KDR antibody on rataortic ring sprouting induced by VEGF.

FIG. 26 shows analysis results for the inhibitory effect of the IgG-typeKDR antibody on angiogenesis induced by VEGF.

FIG. 27 shows analysis results for the inhibitory effect of a 6A6antibody on tumor growth in the HCT116 cell line in a colon cancer mousexenograft model.

FIG. 28 is a photograph of tumors excised after treatment with the 6A6antibody in the colon cancer mouse xenograft model.

FIG. 29 shows analysis results for the inhibitory effect of the 6A6antibody on tumor growth in the A549 cell line in a lung cancer mousexenograft model.

FIG. 30 shows the results of labeling of the IgG-type 6A6 antibody withradioactive isotope iodine.

FIG. 31 shows color images of the IgG-type 6A6 antibody labeled withiodine-123 in a mouse tumor model of chronic myelogenous leukemia.

FIG. 32 is a schematic diagram showing the preparation of light-chainshuffling.

FIG. 33 shows the results of VEGF competition assays of anti-KDRantibodies obtained through light chain shuffling.

FIG. 34 shows the results of VEGF competition assays of anti-KDRantibodies obtained through light chain shuffling.

FIG. 35 shows the results of VEGF competition assays of anti-KDRantibodies obtained through light chain shuffling.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a fully human ScFv(single chain variable fragment) antibody 6A6 (TTAC-0001)-ScFv, whichneutralizes vascular endothelial growth factor.

The 6A6-ScFv was screened in the following manner. First, a library offully human antibodies was constructed, and a cell line expressing afusion protein composed of each of extracellular domains 1-3 ofKDR(VEGFR-2), fused to Fc, was constructed. Anti-KDR-ScFv antibodiesneutralizing KDR were screened from the fully human antibody library inthe cell line using purified recombinant KDR D1-D3-Fc fusion proteins.

The screened ScFv antibodies were expressed and purified in bacteriawith V5 tagging, and human KDR D1-D3-Fc binding assays and VEGFcompetition assays were performed in a ScFv-phage particle state. Also,BIAcore analysis was carried out to measure the ScFv-antibody affinity,and 6A6-ScFv having constantly maintained affinity was obtained andconverted in the form of IgG.

Also, in the present invention, it was confirmed through Westernblotting that 6A6-ScFv inhibited the phosphorylation of an angiogenesissignaling factor ERK in primary cultured HUVEC cells and that thisinhibition was dependent on the concentration of 6A6-ScFv.

In another aspect, the present invention relates to a fully humanantibody 6A6-IgG neutralizing vascular endothelial growth factorreceptor.

FACS analysis revealed that the 6A6-IgG was strongly bound to endogenoushuman KDR, which was expressed on the surface of living HUVEC cells(ATCC, USA), compared to a commercially available IMC-1C11 IgG chimericantibody (Imclone, USA), and that, even when the cells were also treatedwith a competitively binding human VEGF₁₆₅, the 6A6-IgG more effectivelyneutralized KDR, expressed on the surface of the HUVEC cells, comparedto the IMC-1C11 IgG chimeric antibody.

This suggests that the results of the VEGF competition assays in ELISAdiffer from the results indicating that 6A6-IgG and the IMC-1C11 IgGchimeric antibody neutralized KDR at similar levels. That is, the invitro assay results and the in vivo assay results can differ from eachother and there is a limitation in screening highly efficient antibodiesbased on the in vitro assay results.

Also, in the present invention, it could be observed that the 6A6-IgGantibody was more strongly bound to KDR, expressed in the human acutemyeloid eukemia cell line K562 (ATCC, USA), compared to the IMC-1C11IgG.

Moreover, in the present invention, it was confirmed through Westernblotting that 6A6-IgG inhibited the phosphorylation of the angiogenesissignaling factor ERK in primary cultured HUVEC cells and that thisinhibition was increased according t concentration dependent manner of6A6-IgG.

Also, in the present invention, it was observed that the 6A6-IgGaccording to the present invention inhibited the chemotactic motility ofHUVEC cells moving to an environment having VEGF present therein andthat the 6A6-IgG also inhibited the tube formation of HUVEC cells, whichis direct angiogenesis action.

Furthermore, in the present invention, in order to confirm that theinhibitory effect of 6A6-IgG on VEGF effects on HUVEC cells is because6A6-IgG blocks the entrance of VEGF receptors into HUVEC cells,observation with a confocal microscope was performed in an experimentusing a KDR antibody labeled with FITC. As a result, it was obserbedthat the VEGF receptor (KDR) could not enter the cells, when cells weretreated with 6A6-IgG.

Also, through an ex vivo rat aortic ring assay, it was found that, inrat aortic rings treated with 6A6-IgG, vascular sprouting did not occur.Also, angiogenesis was analyzed through a metrigel plug assay byinjecting matrigel subcutaneously into mice.

As a result, in a group treated with VEGF, angiogenesis in plugs wasobserved, but in a group treated with VEGF along with 6A6-IgG,angiogenesis was not observed, suggesting that 6A6-IgG had anangiogenesis inhibitory effect in vivo.

In still another aspect, the present invention relates to variantsobtained by mutating the light chain sequence of 6A6-ScFv through lightchain shuffling.

Through the light chain shuffling, 18 light chain variants of 6A6-ScFvwere obtained, and the light chain shuffling was performed in thefollowing manner.

-   -   (1) In order to prevent 6A6 from being selected again during a        biopanning process, DNA of a 6A6 light chain shuffling library        was treated with a restriction enzyme SpeI having a recognition        site at the CDR3 of 6A6. After the DNA was transfected into ETB        cells, a sub-library was constructed, and KDR affinity and VEGF        competition assays in ELISA were performed to select clones        having excellent KDR neutralizing ability.    -   (2) In a washing step in the biopanning process, the antibody        clones were allowed to compete with soluble KDR to select clones        having excellent KDR neutralizing ability.    -   (3) In a step of allowing phages to bind to the antigen KDR in        the biopanning process, IMC-1121B IgG (ImClone, USA) was also        added in order to select clones having KDR neutralizing ability        which was superior or similar to that of the 1121B IgG.

In still another aspect, the present invention relates to a compositionfor inhibiting angiogenesis and a composition for treating cancer, whichcontain said ScFv or IgG.

As used herein, the term “angiogenesis” includes angiogenesis involvedin tumor growth and metastasis, age-related macular degeneration,rheumatoid arthritis, diabetic retinopathy, psoriasis and chronicinflammation, but the scope of the present invention is not limitedthereto.

In the present invention, said cancer includes, but is not limited to,colon cancer, pancreas cancer, rectal cancer, colorectal cancer,prostate cancer, renal cancer, melanoma, prostate cancer metastasized tobone, ovarian cancer and blood cancer.

The composition of the present invention can be administered by anyroute suitable for a specific molecule. The composition of the presentinvention may be provided to animals, including humans, by any suitablemeans, directly (e.g., locally, such as by injection, subcutaneousinjection or topical administration to a tissue locus) or systemically(e.g., parenterally or orally). Where the composition of the presentinvention is to be provided parenterally, such as by intravenous,subcutaneous, opthalmic, intraperitoneal, intramuscular, buccal, rectal,vaginal, intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracisternal, intracapsular, intranasaladministratiom or by aerosol administration, the composition preferablycomprises part of an aqueous or physiologically compatible fluidsuspension or solution. Thus, the carrier or excipient isphysiologically acceptable so that in addition to delivery of thedesired agent to the subject, the solution does not otherwise adverselyaffect the subject's electrolyte and/or volume balance. The aqueousmedium for the agent thus may comprise normal physiologic saline.

The ScFv or IgG protein of the present invention may be administered fortherapeutic treatments to a cancer patient in an amount sufficient toprevent, inhibit, or reduce the progression of the tumor, e.g., thegrowth, invasiveness, metastases and/or recurrence of the tumor. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the disease and the general state of the patient's ownimmune system.

The dose of the protein according to the present invention is preferably0.01-100 mg/kg, and more preferably 0.1-10 mg/m².

However, the optimal dose will depend upon a disease being treated andthe existence of side effects and can be determined using routineexperimentation. The administration of the antibody may be by periodicbolus injections, or by continuous intravenous or intraperitonealadministration from an external reservoir (for example, from anintravenous bag) or an internal reservoir (for example, from abioerodable implant). Furthermore, the antibody proteins of the presentinvention also may be administered to the intended recipient togetherwith a plurality of different biologically active molecules. However,the optimal combination of fusion protein and other molecules, modes ofadministration, dosages may be determined by routine experimentationwell within the level of skill in the art.

The composition according to the present invention may be used incombination with other therapeutic agents associated with the relevantdisease.

There is synergy when tumors, including human tumors, are treated withthe VEGF receptor antibody in combination with radiation, chemotherapy,an additional receptor antagonist or a combination thereof. In otherwords, the inhibition of tumor growth by a VEGF receptor antagonist isenhanced more than expected when combined with chemotherapeutic agents,radiation, or an additional receptor antagonist or combinations thereof.Synergy may be shown, for example, by greater inhibition of tumor growthwith combined treatment than it would be expected from the additiveeffect of treatment with a VEGF receptor antagonist and achemotherapeutic agent, radiation, or an additional receptor antagonist.Preferably, synergy is demonstrated by remission of the cancer whereremission is not expected from treatment with a combination of a VEGFreceptor antagonist and a chemotherapeutic agent, radiation, or anadditional receptor antagonist.

The VEGF receptor antagonist is administered before, during, or aftercommencing chemotherapy or radiation therapy, as well as any combinationthereof, i.e. before and during, before and after, during and after, orbefore, during, and after commencing the chemotherapy and/or radiationtherapy. For example, when the VEGF receptor antagonist is an antibody,the antibody is typically administered between 1 and 30 days, preferablybetween 3 and 20 days, more preferably between 5 and 12 days beforecommencing radiation therapy and/or chemotherapy.

VEGF Receptor Antibody

In one embodiment, the VEGF receptor antibody binds specifically to anepitope on the extracellular domain of a VEGF receptor. Theextracellular domain of a VEGF receptor is the ligand-binding domain.The ligand-binding domain may be found at either end of the receptor,but is normally found at the amino-terminal end.

Some examples of VEGF receptors include the protein tyrosine kinasereceptors referred to in the literature as Flt-1 (VEGFR-1), KDR andFlk-1 (VEGFR-2). Unless otherwise stated or clearly suggested otherwiseby context, this specification will follow the customary literaturenomenclature of VEGF receptors. KDR will be referred to as the humanform of a VEGF receptor having MW 180 kD (Terman et al., Oncogene,6:1677, 1991). Flk-1 (VEGFR-2) is will be referred to as the murinehomolog of KDR (Matthews et al., PNAS, 88:9026, 1991). Flt-1 (VEGFR-1)is referred to as a form of VEGF receptor different from, but relatedto, the KDR/Flk-1 receptor (Shibuya et al., Oncogene, 5:519, 1990).

Other VEGF receptors include those that can be cross-link with labeledVEGF, or that can be co-immunoprecipitated with KDR. Some known forms ofthese VEGF receptors have molecular weights of approximately 170 kD, 150kD, 130-135 kD, 120-125 kD and 85 kD (Quinn et al., PNAS, 90:7533, 1993;Scher et al., J. Biol. Chem., 271:5761, 1996).

The VEGF receptor is usually bound to a cell, such as an endothelialcell. The VEGF receptor may also be bound to a non-endothelial cell,such as a tumor cell. Alternatively, the VEGF receptor may be free fromthe cell, preferably in soluble form.

The antagonist of the present invention neutralizes VEGF receptors. Inthis specification, neutralizing a receptor means inactivating theintrinsic kinase activity of the receptor to transduce a signal. Areliable assay for VEGF receptor neutralization is the inhibition ofreceptor phosphorylation.

The present invention is not limited by any particular mechanism of VEGFreceptor neutralization. The mechanism caused by one antagonist is notnecessarily the same as that caused by another antagonist. Some possiblemechanisms include preventing binding of the VEGF ligand to theextracellular binding domain of the VEGF receptor, and preventingdimerization or oligomerization of receptors. Other mechanisms cannot,however, be ruled out.

A VEGF receptor (or VEGFR) antibody, in the context of the presentinvention, inhibits activation of the VEGFR subfamily of receptors. Byinhibition of activation of the VEGFR subfamily of receptors is meantany decrease in the activation of the VEGFR. That is, the prevention ofactivation need not completely stop activation of the VEGFR. Moreover,inhibition of VEGFR activation, as defined by the present invention,means inhibition of the VEGFR following interaction of the VEGFRantibody and VEGFR. By association is meant sufficient physical orchemical interaction between the VEGFR and VEGFR antibody which inhibitstyrosine kinase activity of the receptor. One of skill in the art wouldappreciate those examples of such chemical interactions, which includeassociation or bonding, are known in the art and include covalentbonding, ionic bonding, hydrogen bonding, etc. Accordingly, the VEGFRantagonist of the present invention inhibits the tyrosine kinaseactivity of the receptor, which prevents autophosphorylation of thereceptor and phosphorylation of various other proteins involved in theVEGFR signaling pathways. Such pathways, which are involved inregulation of vasculogenesis and angiogenesis, include any of thefollowing: the phospholipase Cy (PLCy) pathway or thephosphatidylinositol 3′ kinase (PI3-K)/Akt and mitogen activated proteinkinase (MAPK) pathway (Larrivee et al., Int. J. Med., 5:447, 2000).

The VEGFR subfamily of receptors is characterized by the presence ofseven immunoglobulin-like loops in the extracellular domain, a singletransmembrane region and a split tyrosine kinase domain in theintracellular region (class III receptor tyrosine kinases). There areseveral known members of the VEGFR subfamily of receptors, examples ofwhich include VEGFR-1, VEGFR-2, and VEGFR-3.

It is generally believed that KDR (VEGFR-2) is the main VEGF signaltransducer that results in endothelial cell proliferation, migration,differentiation, tube formation, increase of vascular permeability, andmaintenance of vascular integrity. VEGFR-1 possesses a much weakerkinase activity, and is unable to generate a mitogenic response whenstimulated by VEGF, although it binds to VEGF with an affinity that isapproximately 10-fold higher than KDR (VEGFR-2). VEGFR-1 has also beenimplicated in VEGF- and placenta growth factor (P1GF)-induced migrationof monocytes and macrophages and production of tissue factor.

As is the case with VEGFR described above, increased VEGFR activationcan result from higher levels of ligand, VEGFR gene amplification,increased transcription of the receptor or mutations that causeunregulated receptor signaling.

In one embodiment of the present invention, the VEGFR antibody inhibitsbinding of VEGFR to its ligand. Binding of a ligand to an external,extracellular domain of VEGFR stimulates receptor dimerization,autophosphorylation of VEGFR, activation of the receptor's internal,cytoplasmic tyrosine kinase domain, and initiation of multiple signaltransduction pathways involved in regulation of vasculogenesis andangiogenesis.

Ligands for VEGFR include VEGF and its homologues P1GF, VEGF-B, VEGF-C,VEGF-D, and VEGF-E. For example, P1GF, which is a dimeric secretedfactor and only binds VEGFR-1, is produced in large amounts by villouscytotrophoblast, sincytiotrophoblast and extravillous trophoblast andhas close amino acid homology to VEGF. Three isoforms exist in humans,P1GF-1, P1GF-2, and P1GF-3. Studies with P1GF-deficient mice demonstratethat this growth factor is not involved in angiogenesis per se, butrather, specifically modulates the angiogenic and permeability effectsof VEGF during pathological situations. Also, VEGF-D is closely relatedto VEGF-C by virtue of the presence of N- and C-terminal extensions thatare not found in other VEGF family members. In adult human tissues,VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, andsmall intestine. Analyses of VEGF-D receptor specificity revealed thatVEGF-D is a ligand for both VEGFR-2 (Flkl) and VEGFR-3 (Flt4) and canactivate these receptors; however, VEGF-D does not bind to VEGFR-1. Inaddition, VEGF-D is a mitogen for endothelial cells.

In another embodiment of the present invention, the VEGFR antibody bindsspecifically to VEGFR. It should be appreciated that the VEGFR antibodycan bind externally to the extracellular portion of VEGFR, which may ormay not inhibit binding of the ligand, or internally to the tyrosinekinase domain. Preferably, the VEGFR antagonist of the present inventionis an antibody, or functional equivalent thereof, specific for VEGFR,details of which are described in more detail below.

In one preferred embodiment, the VEGF receptor antibody bindsspecifically to KDR. Particularly preferred are antigen-binding proteinsthat bind to the extracellular domain of KDR and block binding by one orboth of its ligands, VEGF and P1GF, and/or neutralize VEGF-induced orP1GF-induced activation of KDR.

There also exist various hybridomas that produce VEGFR-2 antibodies. Forexample, a hybridoma cell line producing rat anti-mouse VEGFR-2monoclonal antibody (DC101) was deposited as ATCC HB 11534; a hybridomacell line (M25. 18A1) producing mouse anti-mouse VEGFR-2 monoclonalantibody mAb 25 was deposited as ATCC HB 12152; and a hybridoma cellline (M73.24) producing mouse anti-mouse VEGFR-2 monoclonal antibody mAb73 was deposited as ATCC HB 12153.

In addition, various hybridomas that produce anti-VEGFR-1 antibodiesexist and include, but are not limited to, hybridomas KM1730 (depositedas FERM BP-5697), KM1731 (deposited as FERM BP-5718), KM1732 (depositedas FERM BP-5698), KM1748 (deposited as FERM BP-5699), KM1750 (depositedas FERM BP-5700) disclosed in WO 98/22616, WO 99/59636, AU 5066698 B2,and CA 2328893.

Many other VEGFR antagonists are known in the art. Some examples ofVEGFR antagonists are described in U.S. Pat. Nos. 5,185,438; 5,621,090;5,283,354; 5,270,458; 5,367,057; 5,548,065; 5,747,651; 5,912,133;6,677,434; 6,960,446; 5,840,301; 5,861,499; 6,365,157; 5,955,311;6,365,157; 6,811,779; and WO 2001/66063. U.S. Pat. No. 5,861,301, Termanet al., Oncogene, 6:1677, 1991, WO 94/10202, and WO 95/21865, discloseVEGFR antagonists and, specifically, anti-VEGFR-2 antibodies. Inaddition, anti-VEGFR-2 antibodies are disclosed in U.S. Pat. Nos.6,177,401 and 5,712,395. U.S. Pat. No. 5,981,569 discloses VEGFRantagonists that are organic molecules. Also, bi-specific antibodies(BsAbs), which are antibodies that have two different antigen-bindingspecificities or sites, directed against KDR (VEGFR-2) and VEGFR-1 areknown. Also, Hennequin et al., J. Med. Chem., 42:5369, 1999 disclosescertain quinazolines, quinolines and cinnolines as being useful as VEGFreceptor antagonists (Annie et al., J. Acqu. Immune Defic. Syn. and Hum.Retrovirol., 17: A41, 1998).

Furthermore, assays for the determination of VEGFR antibodies are knownin the art. The VEGFR antibodies of the present invention inhibit thetyrosine kinase activity of VEGFR, which generally involvesphosphorylation events. Accordingly, phosphorylation assays are usefulin determining VEGFR antibodies in the present invention. Some assaysfor tyrosine kinase activity are described in Panek et al., J.Pharmacol. Exp. Thera., 283:1433, 1997 and Batley et al., Life Sci.,62:143, 1998. In addition, methods specific for detection of VEGFRexpression can be utilized.

Antibodies

The antibodies of the present invention may be produced by methods knownin the art (Kohler and Milstein, Nature, 256:495, 1975; Campbell,Monoclonal Antibody Technology, The Production and Characterization ofRodent and Human Hybridomas; Burdon et al., Eds., Laboratory Techniquesin Biochemistry and Molecular Biology, Vol. 13, Elsevier SciencePublishers, Amsterdam, 1985; Huse et al., Science, 246:1275, 1989). Theantibodies of the present invention can be monoclonal or polyclonalantibodies or any other suitable type of an antibody, such as a fragmentor a derivative of an antibody, a single chain variable fragmen(ScFv) ora synthetic homolog of the antibody, provided that the antibody has thesame binding characteristics as, or that has binding characteristicscomparable to, those of the whole antibody. As used herein, unlessotherwise indicated or clear from the context, antibody domains, regionsand fragments follow standard definitions as are well known in the art(Abbas et al., Cellular and Molecular Immunology, W.B. Saunders Company,Philadelphia, Pa., 1991). Preferably, the antibodies of the presentinvention are monoclonal antibodies.

Antibody fragments can be produced by cleaving a whole antibody, or byexpressing DNA that encodes the fragment. Fragments of antibodies may beprepared by methods described in the published literature (Lamoyi etal., J. Immunol. Methods, 56:235, 1983; Parham, J. Immunol., 131:2895,1983). Such fragments may contain one or both of an Fab fragment and anF(ab′)2 fragment. Such fragments may also contain single chain variablefragment antibodies, i.e. scFv, dibodies, or other antibody fragments.Methods of producing such functional equivalents are disclosed in WO93/21319, EP 239,400, WO 89/09622, EP 338,745 and EP 332,424.

Single chain variable fragments (scFv) are polypeptides that consist ofthe variable region of a heavy chain of an antibody linked to thevariable region of a light chain with a short peptide linker). Thus, thescFv comprises the entire antibody-combining site. These chains may beproduced in bacteria, or in eukaryotic cells. A typical example of asingle chain antibody in the present invention is 6A6-ScFv (TTAC-0001).6A6-ScFv was shown to block VEGF-KDR (VEGF-VEGFR-2) interaction andinhibit VEGF-stimulated receptor phosphorylation. This 6A6-ScFv bindsboth to soluble KDR (VEGFR-2) and cell surface-expressed KDR (VEGFR-2)on HUVEC cells and K562 cells. The 6A6-ScFv has a light chain sequenceof SEQ ID NO: 35 and a heavy chain sequence of SEQ ID NO: 36. The6A6-ScFv antibody is a fully human antibody and can be constructed withFab′, F(ab′)2, bivalent ScFv, bivalent recombiant ScFv or human IgGantibodies.

Preferably, although the antibody fragments contain all sixcomplementarity-determining regions (CDRs) of the whole antibody,fragments containing fewer than all of such regions, such as three, fouror five CDRs, may also be functional. If the antibody fragment is tooshort to be immunogenic, it may be conjugated to a carrier molecule.Some suitable carrier molecules include keyhole limpet hemocyanin andbovine serum albumen. Conjugation may be carried out by methods known inthe art.

Antibodies of the present invention also include antibodies whosebinding characteristics can be improved by direct mutation, methods ofaffinity maturation, phage display, or chain shuffling. Affinity andspecificity may be modified or improved by mutating CDRs and screeningfor antigen binding sites having the desired characteristics (Yang etal., J. Mol. Biol., 254:392, 1995). CDRs are mutated in a variety ofways. One way is to randomize individual residues or combinations ofresidues so that in a population of otherwise identical antigen bindingsites, all twenty amino acids are found at particular positions.Alternatively, mutations are induced over a range of CDR residues byerror prone PCR methods ((Hawkins et al., J. Mol. Biol., 226:889, 1992).Phage display vectors containing heavy and light chain variable regiongenes are propagated in mutator strains of E. coli (Low et al., J. Mol.Biol., 250:359, 1996). These methods of mutagenesis are illustrative ofthe many methods known to one of skill in the art.

Antibodies, and particularly monoclonal antibodies, can be produced bymethods known in the art. Examples for production of antibodies include,but are not limited to, production in hybridoma cells and transformationof mammalian cells with DNA encoding the receptor antagonist. Thesemethods are described in various publications (Kohler and Milstein,Nature, 256:495, 1975; Campbell in “Monoclonal Antibody Technology, TheProduction and Chracterization of Rodent and Human Hybridomas” in Burdonet al, Eds., Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 13, Elservier Science Publishers, Amsterdam, 1985; Huse etal., Science, 246:1275, 1989).

Equivalents of antibodies are also prepared by methods known in the art.For example, fragments of antibodies may be prepared enzymatically fromwhole antibodies. Preferably, equivalents of antibodies are preparedfrom DNA encoding such equivalents. DNA encoding fragments of antibodiesmay be prepared by deleting all but the desired portion of the DNA thatencodes the full-length antibody. DNA encoding chimerized antibodies maybe prepared by recombining DNA encoding human constant regions, derivedsubstantially or exclusively from the corresponding human antibodyregions, and DNA encoding variable regions, derived substantially orexclusively from the sequence of the variable region of a mammal otherthan a human. DNA encoding humanized antibodies may be prepared byrecombining DNA encoding constant regions and variable regions otherthan the complementarity determining regions (CDRs), derivedsubstantially or exclusively from the corresponding human antibodyregions, and DNA encoding CDRs, derived substantially or exclusivelyfrom a mammal other than a human.

Suitable sources of DNA molecules that encode fragments of antibodiesinclude cells, such as hybridomas, that express the full-lengthantibody. The fragments may be used by themselves as antibodyequivalents, or may be recombined into equivalents, as described above.The DNA deletions and recombinations described in this section may becarried out by known methods, such as those described in the publishedpatent applications listed above in the section entitled “FunctionalEquivalents of Antibodies” and/or other standard recombinant DNAtechniques, such as those described below.

Preferred host cells for transformation of vectors and expression of theantibodies of the present invention are mammalian cells, e.g., COS-7cells, Chinese hamster ovary (CHO) cells, and cell lines of lymphoidorigin such as lymphoma, myeloma, or hybridoma cells. Other eukaryotichost, such as yeasts, can be alternatively used. For example, mousefetal liver stromal cell line 2018 binds to APtag-Flk 1 and APtag-Flk-2fusion proteins, i.e., contains ligands of VEGFR-2 and Flk-2 (ATCC,Manassas, Va., CRL 10907), human fetal spleen cell line Fsp 62891contains Flk-2 ligand (ATCC CRL 10935), and human stromal fetal thymuscell line, F. thy 62891, contains VEGFR-2 ligand (ATCC CRL 10936).

As used herein, the term “vector” means any nucleic acid comprising acompetent nucleotide sequence to be incorporated into a host cell and tobe recombined with and integrated into the host cell genome, or toreplicate autonomously as an episome. Such vectors include linearnucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectorsand the like. Examples of a viral vector include, but is not limited to,a retrovirus, an adenovirus and an adeno-associated virus.

As used herein, the term “gene expression” or “expression of a targetprotein” is understood to mean the transcription of a DNA sequence, thetranslation of an mRNA transcript and the secretion of an Fc fusionprotein product.

In the present invention, suitable host cells can be transformed ortransfected with DNA and can be used to express and/or secrete targetproteins. Preferred host cells for use in the present invention includeimmortalized hybridoma cells, NS/O myeloma cells, 293 cells, Chinesehamster ovary (CHO) cells, HELA cells and COS cells.

The transformed host cells are cultured by methods known in the art in aliquid medium containing assimilable sources of carbon (carbohydratessuch as glucose or lactose), nitrogen (amino acids, peptides, proteinsor their degradation products such as peptones, ammonium salts or thelike), and inorganic salts (sulfates, phosphates and/or carbonates ofsodium, potassium, magnesium and calcium). The medium additionallycontains, for example, growth-promoting substances, such as traceelements, for example iron, zinc, manganese and the like.

Where it is desired to express a gene construct in yeast, a suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 (Stinchcomb et al., Nature, 282:39, 1979; Kingsman et al.,Gene, 7:141, 1979). The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC 44076 or PEP4-1 (Jones, Genetics, 85:12, 1977). Thepresence of the trpl lesion in the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC20622 or 38626) are complemented by known plasmids bearing the Leu2gene.

Alternatively, the DNA encoding the receptor antagonist can be clonedinto vectors derived from viruses such as adenovirus, adeno-associatedvirus, herpesvirus, retrovirus or lentivirus. Gene expression iscontrolled by inducible or uninducible regulatory sequences.

Briefly, a suitable source of cells containing nucleic acid moleculesthat express the desired DNA, such as an antibody, antibody equivalentor VEGF receptor, is selected. Total RNA is prepared by standardprocedures from a suitable source. The total RNA is used to direct cDNAsynthesis. Standard methods for isolating RNA and synthesizing cDNA areprovided in standard manuals of molecular biology such as, for example,those described above.

The cDNA may be amplified by known methods. For example, the cDNA may beused as a template for amplification by polymerase chain reaction (PCR)(Saiki et al., Science, 239:487, 1988; U.S. Pat. No. 4,683,195). Thesequences of the oligonucleotide primers for the PCR amplification arederived from the known sequence to be amplified. The oligonucleotidesare synthesized by methods known in the art (Caruthers, Science,230:281, 1985).

A mixture of upstream and downstream oligonucleotides is used in the PCRamplification. The conditions are optimized for each particular primerpair according to standard procedures. The PCR product is analyzed, forexample, by electrophoresis for cDNA having the correct size,corresponding to the sequence between the primers. Alternatively, thecoding region may be amplified in two or more overlapping fragments. Theoverlapping fragments are designed to include a restriction sitepermitting the assembly of the intact cDNA from the fragments.

In order to isolate the entire protein-coding regions for the VEGFreceptors, for example, the upstream PCR oligonucleotide primer iscomplementary to the sequence at the 5′ end, preferably encompassing theATG start codon and at least 5-10 nucleotides upstream of the startcodon. The downstream PCR oligonucleotide primer is complementary to thesequence at the 3′ end of the desired DNA sequence. The desired DNAsequence preferably encodes the entire extracellular portion of the VEGFreceptor, and optionally encodes all or part of the transmembraneregion, and/or all or part of the intracellular region, including thestop codon.

The DNA to be amplified, such as that encoding antibodies, antibodyequivalents, or VEGF receptors, may also be replicated in a wide varietyof cloning vectors in a wide variety of host cells. The host cell may beprokaryotic or eukaryotic.

The vector into which the DNA is spliced may comprise segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Some suitableprokaryotic cloning vectors include plasmids derived from E. coli, suchas colE1, pCR1, pBR322, pMB9, pKSM, and RP4. Prokaryotic vectors alsoinclude derivatives of phage DNA such as M13 and other filamentoussingle-stranded DNA phages. A preferred vector for cloning nucleic acidencoding the VEGF receptor is the Baculovirus vector.

The vector containing the DNA to be expressed is transfected into asuitable host cell. The host cell is maintained in an appropriateculture medium, and subjected to conditions under which the cells andthe vector replicate. The vector may be recovered from the cell. The DNAto be expressed may be recovered from the vector.

The DNA to be expressed, such as that encoding antibodies, antibodyequivalents, or receptors, may be inserted into a suitable expressionvector and expressed in a suitable prokaryotic or eucaryotic host cell.

For example, the DNA inserted into a host cell may encode the entireextracellular portion of the VEGF receptor, or a soluble fragment of theextracellular portion of the VEGF receptor. The extracellular portion ofthe VEGF receptor encoded by the DNA is optionally attached at either,or both, the 5′ end or the 3′ end to additional amino acid sequences.The additional amino acid sequences may be attached to the VEGF receptorextracellular region, such as the leader sequence, the transmembraneregion and/or the intracellular region of the VEGF receptor. Theadditional amino acid sequences may also be sequences not attached tothe VEGF receptor in nature. Preferably, such additional amino acidsequences serve a particular purpose, such as to improve expressionlevels, secretion, solubility, or immunogenicity.

Vectors for expressing proteins in bacteria, especially E. coli, areknown (Dieckmann and Tzagoloff, J. Biol. Chem., 260:1513, 1985). Thesevectors contain DNA sequences that encode anthranilate synthetase (TrpE)followed by a polylinker at the carboxy terminus. Other expressionvector systems are based on beta-galactosidase (pEX); lambda PL; maltosebinding protein (pMAL); and glutathione S-transferase (pGST) (Gene,67:31, 1988; Peptide Research, 3:167, 1990).

Suitable vectors for expression in mammalian cells are also known. Suchvectors include well-known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences and shuttle vectors derived fromcombination of functional mammalian vectors, such as those describedabove, and functional plasmids and phage DNA.

Additional vectors for expression of eukaryotic cells are known in theart (Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1:327, 1982;Subramani et al, Mol. Cell. Biol., 1:854, 1981; Kaufmann and Sharp, J.Mol. Biol., 159:601, 1982; Kaufinann and Sharp, Mol. Cell. Biol., 1982;Scahill et al., PNAS, 80:4654, 1983; Urlaub and Chasin, PNAS, 77:4216,1980). The expression vectors useful in the present invention contain atleast one expression control sequence that is operatively linked to theDNA sequence or fragment to be expressed. The control sequence isinserted in the vector in order to control and to regulate theexpression of the cloned DNA sequence. Examples of useful expressioncontrol sequences include the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the control region of fd coat protein, the glycolytic promotersof yeast, e.g., the promoter for 3-phosphoglycerate kinase, thepromoters of yeast acid phosphatase, e.g., PhoS, the promoters of theyeast alpha-mating factors, and promoters derived from polyoma,adenovirus, retrovirus, and simian virus, e.g., the early and latepromoters of SV40, and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells and their viruses.

Vectors containing the control signals and DNA to be expressed, such asthat encoding antibodies, antibody equivalents, or VEGF receptors, areinserted into a host cell for expression. Some useful expression hostcells include well-known prokaryotic and eukaryotic cells. Some suitableprokaryotic hosts include, for example, E. coli, such as E. coli SG-936,E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coliDHI, and E. coli MRCI, Pseudomonas, Bacillus, such as Bacillus subtilis,and Streptomyces. Suitable eukaryotic cells include yeast and otherfungi, insect, animal cells, such as COS cells and CHO cells, humancells and plant cells in tissue culture.

Following expression in a host cell maintained in a suitable medium, thepolypeptide or peptide to be expressed, such as antibodies, antibodyequivalents, or VEGF receptors, may be isolated from the medium, andpurified by methods known in the art. If the polypeptide or peptide isnot secreted into the culture medium, the host cells are lysed prior toisolation and purification.

In addition, the antibodies of the invention may be prepared byimmunizing a mammal with a soluble receptor. The soluble receptorsthemselves may be used as immunogens, or may be attached to a carrierprotein or to other objects, such as beads, i.e., sepharose beads. Afterthe mammal has produced antibodies, a mixture of antibody-producingcells, such as the splenocytes, is isolated. Monoclonal antibodies maybe produced by isolating individual antibody-producing cells from themixture and making the cells immortal by, for example, fusing them withtumor cells, such as myeloma cells. The resulting hybridomas arepreserved in culture, and express monoclonal antibodies, which areharvested from the culture medium.

The antibodies may also be prepared from receptors bound to the surfaceof cells that express the specific receptor of interest. The cell towhich the receptors are bound may be a cell that naturally expresses thereceptor, such as a vascular endothelial cell for VEGFR. Alternatively,the cell to which the receptor is bound may be a cell into which the DNAencoding the receptor has been transfected, such as 3T3 cells, whichhave been transfected with VEGFR.

A receptor may be used as an immunogen to raise an antibody of thepresent invention. The receptor peptide may be obtained from naturalsources, such as from cells that express the receptors. For example, theVEGF receptor peptide may be obtained from vascular endothelial cells.Alternatively, synthetic receptor peptides may be prepared usingcommercially available machines. In such an embodiment, the VEGFreceptor amino acid sequence can be provided through the publishedliteratures (Shibuya et al., Oncogene, 5:519, 1990; PCT/US92/01300;Terman et al., Oncogene, 6:1677, 1991; Matthews et al., PNAS, 88:9026,1991).

As an alternative, DNA encoding a receptor, such as a cDNA or a fragmentthereof, is cloned and expressed, and the resulting polypeptide isrecovered and thus it may be used as an immunogen to raise an antibodyof the present invention. For example, in order to prepare the VEGFreceptors against which the antibodies are made, nucleic acid moleculesthat encode the VEGF receptors of the present invention, or portionsthereof, especially the extracellular portions thereof, may be insertedinto known vectors for expression in host cells using standardrecombinant DNA techniques, such as those described below. Suitablesources of such nucleic acid molecules include cells that express VEGFreceptors, i.e., vascular endothelial cells.

The antibody may be prepared in any mammal; suitable mammals other thanhuman include, for example, a rabbit, rat, mouse, horse, goat, orprimate. Mice are frequently used to prepare monoclonal antibodies. Theantibody may be a member of one of the following immunoglobulin classes:IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof, and preferablyis an IgG1 antibody. The antibodies of the present invention and theirfunctional equivalents may be or may combine members of any of theimmunoglobulin classes.

Neutralization of VEGF Activation of VEGF Receptors

Neutralization of activation of a VEGF receptor in a sample ofendothelial or non-endothelial cells, such as tumor cells, may beperformed in vitro or in vivo. Neutralizing VEGF activation of a VEGFreceptor in a sample of VEGF-receptor expressing cells comprisescontacting the cells with the antibody of the present invention. Thecells are contacted in vitro with the antibody, before, simultaneouslywith, or after, adding VEGF to the cell sample.

In vivo, the antibody of the present invention is contacted with a VEGFreceptor by administration to a mammal. Methods of administration to amammal include, for example, oral, intravenous, intraperitoneal,subcutaneous, or intramuscular administration.

This in vivo neutralization method is useful for inhibiting angiogenesisin a mammal. Angiogenesis inhibition is a useful therapeutic method,such as for preventing or inhibiting angiogenesis associated withpathological conditions such as tumor growth. Accordingly, the antibodyof the present invention is an anti-angiogenic and anti-tumorimmunotherapeutic agent.

As used herein the term “mammal” means any mammal. Some examples ofmammals include pet animals, such as dogs and cats; farm animals, suchas pigs, cattle, sheep, and goats; laboratory animals, such as mice andrats; primates, such as monkeys, apes, and chimpanzees; and humans.

VEGF receptors are found on some non-endothelial cells, such as tumorcells, indicating the unexpected presence of an autocrine and/orparacrine loop in these cells. The antagonists, e.g., the antibodies, ofthis invention are useful in neutralizing the activity of VEGF receptorson such cells, thereby blocking the autocrine and/or paracrine loop, andinhibiting tumor growth. The methods of inhibiting angiogenesis and ofinhibiting pathological conditions such as tumor growth in a mammalcomprise administering an effective amount of any one of the inventiveantagonists, e.g., antibodies, including any of the functionalequivalents thereof, systemically to a mammal, or directly to a tumorwithin the mammal. The mammal is preferably human. This method iseffective for treating subjects with both solid tumors, preferablyhighly vascular tumors, and non-solid tumors.

The inhibition or reduction of tumor growth includes the prevention orinhibition of the progression of a tumor, including cancerous andnoncancerous tumors. The progression of a tumor includes theinvasiveness, metastasis, recurrence and increase in size of the tumor.The inhibition or reduction of tumor growth also includes thedestruction of a tumor.

All types of tumors may be treated by the methods of the presentinvention. The tumors may be solid or non-solid.

Some examples of solid tumors that can be treated with the antagonistsof the present invention include carcinomas, sarcomas, blastomas orgliomas. Some examples of such tumors include epidermoid tumors,squamous tumors, such as head and neck tumors, colorectal tumors,prostate tumors, breast tumors, lung tumors, including small cell andnon-small cell lung tumors, pancreatic tumors, thyroid tumors, ovariantumors, and liver tumors. Other examples include Kapos's sarcoma, CNSneoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas andcerebral metastases, melanoma, gastrointestinal and renal carcinomas andsarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastomamultiforme, and leiomyosarcoma. Examples of vascularized skin cancersfor which the antagonists of this invention are effective includesquamous cell carcinoma, basal cell carcinoma and skin cancers that canbe treated by suppressing the growth of malignant keratinocytes, such ashuman malignant keratinocytes.

Some examples of non-solid tumors include leukemias, multiple myelomasand lymphomas. Some examples of leukemias include acute myelocyticleukemia (AML), chronic myelocytic leukemia (CML), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocyticleukemia or monocytic leukemia. Some examples of lymphomas includelymphomas associated with Hodgkin's disease and non-Hodgkin's disease.

Preventing or inhibiting angiogenesis is also useful to treatnon-neoplastic pathologic conditions characterized by excessiveangiogenesis, such as neovascular glaucoma, proliferative retinopathyincluding proliferative diabetic retinopathy, arthritis, maculardegeneration, hemangiomas, angiofibromas, and psoriasis.

Using Inventive Antibodies to Isolate and Purify VEGF Receptor

The antagonists of the present invention may be used to isolate andpurify the VEGF receptor using conventional methods such as affinitychromatography (Dean et al., Affinity Chromatography: A PracticalApproach, IRL Press, Arlington, Va., 1985). Other methods well known inthe art include magnetic separation with antibody-coated magnetic beads,“panning” with an antibody attached to a solid matrix, and flowcytometry (FACS).

The source of the VEGF receptor is typically vascular cells, andespecially vascular endothelial cells, that express the VEGF receptor.Suitable sources of vascular endothelial cells are blood vessels, suchas umbilical cord blood cells, especially, human umbilical cord vascularendothelial cells (HUVEC).

The VEGF receptors may be used as a starting material to produce othermaterials, such as antigens for making additional monoclonal andpolyclonal antibodies that recognize and bind to the VEGF receptor orother antigens on the surface of VEGF-expressing cells.

Using Inventive Antibodies to Isolate and Purify KDR Positive TumorCells

The antibodies of the present invention may be used to isolate andpurify Flk-1 KDR (VEGFR-2) positive tumor cells, i.e., tumor cellsexpressing KDR, using conventional methods such as affinitychromatography (Dean, P. D. G. et al., Affinity Chromatography: APractical Approach, IRL Press, Arlington, Va., 1985). Other methods wellknown in the art include magnetic separation with antibody-coatedmagnetic beads, cytotoxic agents, such as complement, conjugated to theantibody, “panning” with an antibody attached to a solid matrix, andflow cytometry (FACS).

Monitoring Levels of VEGF and VEGF Receptors In Vitro or In Vivo

The antibodies of the present invention may be used to monitor thelevels of VEGF or VEGF receptors in vitro or in vivo in biologicalsamples using standard assays and methods known in the art. Someexamples of biological samples include bodily fluids, such as blood.Standard assays involve, for example, labeling the antibodies andconducting standard immunoassays, such as radioimmunoassays, as is wellknow in the art.

Standard recombinant DNA techniques useful in carrying out the presentinvention are described in the literature (Sambrook et al., “MolecularCloning, “Second Edition, Cold Spring Harbor Laboratory Press, 1987;Ausubel et al, (Eds) “Current Protocols in Molecular Biology, “GreenPublishing Associates/Wiley-Interscience, New York, 1990).

Administration

The receptor antibodies of the present invention can be administered fortherapeutic treatments to a patient suffering from a tumor in an amountsufficient to prevent, inhibit, or reduce the progression of the tumor,e.g., the growth, invasiveness, metastases and/or recurrence of thetumor. An amount adequate to accomplish this purpose is defined as atherapeutically effective dose. Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's own immune system. Dosing schedules will also vary dependingon the disease state and status of the patient, and will typically rangefrom a single bolus dosage or continuous infusion to multipleadministrations per day (e.g., every 4-6 hours), or as indicated by thetreating physician and the patient's condition. It should be noted,however, that the present invention is not limited to any particulardose.

The present invention can be used to treat any suitable tumor,including, for example, tumors of the breast, heart, lung, smallintestine, colon, spleen, kidney, bladder, head and neck, ovary,prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus,uterus, testicles, cervix or liver. Preferably, the methods of thepresent invention are used when the tumor is a tumor of the colon orwhen the tumor is a non-small cell lung carcinoma (NSCLC).

Furthermore, the tumors of the present invention preferably haveaberrant expression or signaling of VEGFR. Enhanced signaling by VEGFRhas been observed in many different human cancers. High levels ofVEGFR-2 are expressed by endothelial cells that infiltrate gliomas(Plate et al., Nature, 359:845, 1992). VEGFR-2 levels are specificallyupregulated by VEGF produced by human glioblastomas (Plate et al.,Cancer Res., 53:5822, 1993). The finding of high levels of VEGFR-2expression in glioblastoma associated endothelial cells (GAEC) indicatesthat receptor activity is probably induced during tumor formation sinceVEGFR-2 transcripts are barely detectable in normal brain endothelialcells. This upregulation is confined to the vascular endothelial cellsin close proximity to the tumor.

The present invention is useful for inhibition or reduction of tumorgrowth. By inhibition or reduction of tumor growth is meant prevention,inhibition, or reduction of the progression of the tumor, e.g, thegrowth, invasiveness, metastases and/or recurrence of the tumor. Inaddition, the present invention can also be useful in treating anangiogenic condition, such as atherosclerosis, arthritis, maculardegeneration and psoriasis. The identification of those patients thathave conditions for which the present invention is useful is well withinthe ability and knowledge of one skilled in the art.

In the present invention, any suitable method or route can be used toadminister the VEGFR antibodies, for example, oral, intravenous,intraperitoneal, subcutaneous, or intramuscular administration. The doseof antagonist administered depends on numerous factors, including, forexample, the type of antibodies, the type and severity of tumor to betreated and the route of administration of the antibodies. It should beemphasized, however, that the present invention is not limited to anyparticular method or route of administration.

In one alternative embodiment, the VEGFR antagonist and can beadministered in combination with one or more antineoplastic agents (U.S.Pat. No. 6,217,866). Any suitable antineoplastic agent can be used, suchas a chemotherapeutic agent or radiation. Examples of chemotherapeuticagents include, but are not limited to, cisplatin, doxorubicin,paclitaxel, irinotecan (CPT-11), topotecan or a combination thereof.When the antineoplastic agent is radiation, the source of the radiationcan be either external (external beam radiation therapy-EBRT) orinternal (brachytherapy-BT) to the patient being treated. The dose ofantineoplastic agent administered depends on numerous factors,including, for example, the type of agent, the type and severity oftumor being treated and the route of administration of the agent. Itshould be emphasized, however, that the present invention is not limitedto any particular dose.

In an additional alternative embodiment, the VEGFR antibody of thepresent invention can be administered in combination with one or moresuitable adjuvants, such as, for example, cytokines (for example, IL-10and IL-13) or other immune stimulators. See, for example, Larrivee etal., supra. It should be appreciated, however, that administration ofonly the VEGFR antagonist is sufficient to prevent, inhibit, or reducethe progression of the tumor in a therapeutically effective manner.

In addition, the VEGFR antibody can be administered as a ligandconjugate, which binds specifically to the receptor and delivers atoxic, lethal payload following ligand-toxin internalization. Conjugatesbetween toxins and the receptors were designed with the aim ofdeveloping toxic agents specific for EGFR- or VEGFR-overexpressing tumorcells while minimizing nonspecific toxicity. For example, a conjugatecomposed of EGF and Pseudomonas endotoxin (PE) was shown to be toxictoward EGFR-expressing HeLa cells in vitro. Various agents, includingthioridazine and adenovirus, can enhance cellular uptake of theconjugate, as well as increase the cytotoxicity of the conjugate.

It is understood that the VEGFR antibodies of the present invention,where used in a mammal for the purpose of prophylaxis or treatment, willbe administered in the form of a composition additionally comprising apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the binding proteins. The compositions for theinjection may, as is well known in the art, be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the mammal.

The VEGFR antibodies of the present invention may be in a variety offorms. These include, for example, solid, semi-solid and liquid dosageforms, such as tablets, pills, powders, liquid solutions, dispersions orsuspensions, liposomes, suppositories, injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application.

Such antibodies can be prepared in a manner well known in thepharmaceutical art. In making the composition, the active ingredientwill usually be mixed with a carrier, or diluted by a carrier, and/orenclosed within a carrier which may, for example, be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it may be a solid, semi-solid, or liquid material, which actsas a vehicle, excipient or medium for the active ingredient. Thus, thecomposition may be in the form of tablets, lozenges, sachets, cachets,elixirs, suspensions, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, injectionsolutions, suspensions, sterile packaged powders, and a topical patch.

Radiation

The source of radiation, used in combination with a VEGF receptorantagonist, can be either external or internal to the patient beingtreated. When the source is external to the patient, the therapy isknown as external beam radiation therapy (EBRT). When the source ofradiation is internal to the patient, the treatment is calledbrachytherapy (BT).

The radiation is administered in accordance with well known standardtechniques using standard equipment manufactured for this purpose, suchas AECL Theratron and Varian Clinac. The dose of radiation depends onnumerous factors as is well known in the art. Such factors include theorgan being treated, the healthy organs in the path of the radiationthat might inadvertently be adversely affected, the tolerance of thepatient to radiation therapy, and the area of the body in need oftreatment. The dose will typically be between 1 and 100 Gy, and moreparticularly between 2 and 80 Gy. Some doses that have been reportedinclude 35 Gy to the spinal cord, 15 Gy to the kidneys, 20 Gy to theliver, and 65-80 Gy to the prostate. It should be emphasized, however,that the present invention is not limited to any particular dose. Thedose will be determined by treating physician in accordance withparticular factors in a given situation, including the factors mentionedabove.

The distance between the source of the external radiation and the pointof entry into the patient may be any distance that represents anacceptable balance between killing target cells and minimizing sideeffects. Typically, the source of the external radiation is between 70cm and 100 cm from the point of entry into the patient.

Brachytherapy is generally carried out by placing the source ofradiation in the patient. Typically, the source of radiation is placedapproximately 0-3 cm from the tissue being treated. Known techniquesinclude interstitial, intercavitary, and surface brachytherapy. Theradioactive seeds can be implanted permanently or temporarily. Sometypical radioactive atoms that have been used in permanent implantsinclude iodine-125 and radon. Some typical radioactive atoms that havebeen used in temporary implants include radium, cesium-137, andiridium-192. Some additional radioactive atoms that have been used inbrachytherapy include americium-241 and gold-198.

The dose of radiation for brachytherapy can be the same as thatmentioned above for external beam radiation therapy. In addition to thefactors mentioned above for determining the dose of external beamradiation therapy, the nature of the radioactive atom used is also takeninto account in determining the dose of brachytherapy.

Chemotherapy

Chemotherapeutic agents include all chemical compounds that areeffective in inhibiting tumor growth. The administration ofchemotherapeutic agents can be accomplished in a variety of waysincluding systemically by the parenteral and enteral routes. In oneembodiment, the VEGF receptor antagonist and the chemotherapeutic agentare administered as separate molecules. In another embodiment, the VEGFreceptor antagonist is attached, for example, by conjugation, to achemotherapeutic agent.

Examples of chemotherapeutic agents include alkylating agents, forexample, nitrogen mustards, ethyleneimine compounds and alkylsulphonates; antimetabolites, for example, folic acid, purine orpyrimidine antagonists; mitotic inhibitors, for example, vinca alkaloidsand derivatives of podophyllotoxin; cytotoxic antibiotics; compoundsthat damage or interfere with DNA expression.

Additionally, chemotherapeutic agents include antibodies, biologicalmolecules and small molecules, as described herein. Particular examplesof chemotherapeutic agents or chemotherapy include cisplatin,dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard),streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNE),doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin,cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine,vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere),aldesleukin, asparaginase, busulfan, carboplatin, cladribine,dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide,interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine,plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin,streptozocin, tamoxifen, teniposide, testolactone, thioguanine,thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol andcombinations thereof.

The present invention also includes kits for inhibiting tumor growthcomprising a therapeutically effective amount of an EGFR antagonist anda therapeutically effective amount of a VEGFR antagonist. The EGFR orVEGFR antagonist of the inventive kits can be any suitable antagonist,examples of which have been described above. Preferably, the EGFRantagonist of the kit comprises an antibody or functional equivalentthereof, specific for EGFR. Alternatively, and also preferably, the EGFRantagonist of the kit comprises a small molecule specific for EGFR. TheVEGFR antagonist of the kit preferably comprises an antibody orfunctional equivalent thereof, specific for VEGFR. Alternatively, theVEGFR antagonist of the kit preferably comprises a small moleculespecific for VEGFR. In addition, the kits of the present invention canfurther comprise an antineoplastic agent. Examples of suitableantineoplastic agents in the context of the present invention have beendescribed herein. The kits of the present invention can further comprisean adjuvant, examples of which have also been described above.

Accordingly, the receptor antibodies of the present invention can beused in vivo and in vitro for investigative, diagnostic, prophylactic,or treatment methods, which are well known in the art. Of course, it isto be understood and expected that variations in the principles ofinvention herein disclosed can be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

EXAMPLES

Hereinafter, the present invention will be described in further detail.It is to be understood, however, that these examples are illustrativepurpose only and are not to be construed to limit the scope of thepresent invention.

Example 1 Establishment of KDR-Fc Secreting Cell Line

The gene corresponding to the extracellular domains (ECDs) 1-3 of theKDR gene (accession no. AF063658 in GenBank) was amplified from a humanplacental cDNA library (Clonetech, USA). The amplification was carriedout using the primers KDR 1F (SEQ ID NO: 21) and KDR 3R (SEQ ID NO: 22)having BamHI and NheI digestion sites respectively.

SEQ ID NO: 21: 5′-CGC GGATCC ATGGAG AGCAA-3′ SEQ ID NO: 22:5′-CCGCTAGC TTTTTCATGGACCCTGACA-3′

To produce a KDR(ECD1-3)-Fc chimeric protein, a pcDNA3-BACE-Fc vector(Korean Patent Publication 10-2005-0032177) composed of a BACE-Fcprotein gene inserted into a pcDNA3 vector (Invitrogen, USA) wasdigested with BamHI and NheI, and then ligated with the PCR fragmentdigested with the same restriction enzymes. The Fc domain was amplifiedby PCR using the primers ThFc-F (SEQ ID NO: 23) and MycFc-R (SEQ ID NO:24) so as to have a thrombin digestion site and a myc tag, and theamplified fragment was ligated with the vector using NheI and XhoIsites, thus constructing pcDNA3-KDR D123tFcm.

SEQ ID NO: 23: 5′-CCGCTAGCAGCGGCCTGGTGCCGCGCGGCAG CGACAAAACTCAC-3′:SEQ ID NO: 24: 5′-GGCTCGAGTCACAGGTCTTCCTCAGAGATCAGC TTCTGCTCTTACCCGGAGAC-3′

The pcDNA3-KDR D123tFcm consists of a base sequence encoding amino acidresidues 1-327 comprising the secretion signal sequence andextracellular domain of human KDR, a base sequence encoding a thrombinrecognition site (SSGLVPRGS), a base sequence encoding 227 amino acidscorresponding to the Fc domain of human immunoglobulin G (hIgG), and abase sequence (EQKLISEEDL) encoding the myc tag (FIG. 1).

For epitope mapping of antibodies, KDR (ECD1-2)-Fc (amino acid residues1-222) and KDR(ECD2-3)-Fc (amino acid residues 1-327 (424-116)) wereprepared. To clone KDR (ECD1-2), the prepared sequence was amplified byPCR using a primer KDR 1F (5′-CGC GGATCC ATGGAG AGCAA: SEQ ID NO: 25)and a primer KDR 12R (5′-CTA GCTAGC CCTAT ACCCT ACAAC GACA-3′: SEQ IDNO: 26), and then the PCR amplified fragment was inserted intopcDNA3-KDR D123tFcm digested with BamHI and NheI, thus preparingpcDNA3-KDR D12tFcm. To clone KDR (ECD2-3), a PCR fragment of the primerKDR 1F and the primer KDR 23SR (SEQ ID NO: 26) and a PCR fragment of theprimer KDR 23SF (SEQ ID NO: 27) and the primer KDR 23R (SEQ ID NO: 28)were amplified by overlap PCR. The resulting PCR fragment was insertedinto pcDNA3-KDR D123tFcm using BamHI and NheI sites, thus preparingpcDNA3-KDR D23tFcm (FIG. 2).

SEQ ID NO: 26: 5′-ACA TAACCC ACAG AGGCG GCCCGGG TCTCCA-3′ SEQ ID NO: 27:5′-GACCCGGGCCGCCTCTGTGGGTTATGTTCAAGATTACAGA-3′ SEQ ID NO: 28:5′-CTA GCTAGC TTTTTCA TGGACCCTGACA-3′

To produce a KDR(ECD)-Fc chimeric protein, the above-prepared pcDNA3-KDRD123tFcm vector was transfected into CHO-DG44 cells (Aprogen, Korea),and the cells were cultured in α-MEM(GibCo, USA), containing 10% dFBS(Gibco, USA) and 500 μg/ml G418 (geneticin; Sigma, USA). To optimize theexpression of the KDR(ECD)-Fc chimeric protein, the cells were culturedin CHO—SFM2 medium (Gibco) in the presence of MTX (methotrexate, Sigma),while the MTX concentration was increased. As a result, it was confirmedthat the protein was optimally expressed at 700 nM MTX.

The produced protein was purified using protein A affinitychromatography (protein A-Sepharose, GE healthcare) and size exclusionchromatography (Hiload superdex 200, GE healthcare) and stored in 10 mMphosphate buffer (pH 7.0) containing 150 mM NaCl. FIG. 3 shows theresults of SDS-PAGE of KDR(ECD1-3)-Fc purified according to the abovemethod.

Example 2 Preparation of Complete Human (Naïve) Single Chain Antibody(ScFv) Phage Display Library

Total RNA was obtained from five healthy bone-marrow donors using TR1reagents (Sigma), and based on the total RNA, mRNA was purified using anmRNA purification kit (oligotex mRNA preparation kit, Qiagen, USA). ThemRNA was treated using an RT-PCR system (ThermoScript RT-PCR system,Gibco-BRL, USA) to obtain cDNA. To obtain a VH gene, each of a V genefragment and a DJ fragment was amplified using the primers shown inTable 1, and each of the amplified DNA fragments was amplified by 2^(nd)PCR using primers (SEQ ID NOS: 29-61) having SfiI restriction enzymesites at the 5′ end and the 3′ end.

TABLE 1 Primer sequence for amplifying VH and DJ gene fragments SEQ IDNO: VH gene-forward H05 GARGTGCAGCTGGTGGAGTC 29 H06 CAGSTGCAGCTGCAGGAGTC30 H08 CAGGTACAGCTGCAGCAGTC 31 H09 CAGRTGCAGCTGGTGCAGTCTGGGG 32 H11GAGGTGCAGCTGGTGCAGTCTGGAGCA 33 H12 CAGGTTCAGCTGGTGCAGTCTGGAG 34 H13CAGGTTCAGCTGGTGCAGTCTGGGG 35 H14 CAGGTCCAGCTGGTACAGTCTGGGG 36 H15CAGGTCACCTTGAAGGAGTCTGGTCCTGT 37 H16 CAGATCACCTTGAAGGAGTCTGGTCCTAC 38H17 CAGGTCACCTTGAGGGAGTCTGGTCCTGC 39 H25 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTG40 H32 CAGGTGCAGCTACAGCAGTGGGGCG 41 VH gene-back H210AATACACGGCCGTGTCCTCAGATC 42 H210L AATACACGGCCGTGTCCTCAGATCTCAGGCT 43GCTCAGCTCCATGTAGGCTGAG H211 AGCTCCATGTAGGCTGTGTCT 44 H212AGCTCCATGTAGGCTGTGCTCATAGACC 45 H213 AGCTCCATGTAGGCTGTGCTTGTGGACA 46H214 AGCTCCATGTAGGCTGTGCTTATGGAG 47 H220 AAGGACCACCTGCTTTTGGAGG 48 H230AATACACGGCCGTGTCCTCGGCTCTCAGACT 49 GTTCATT H240 AATACACGGCCTGTCCACGGCGG50 H250 AATACATGGCGGTGTCCGAGGCCT 51 DJ gene-forward CDR3-1GATCTGAGGACACGGCCGTGTATTACTGT 52 CDR3-2 CCTCCAAAAGCCAGGTGGTCCTT 53CDR3-3 GAGCCGAGGACACGGCCGTGTATTACTGT 54 CDR3-4CCGCCGTGGACACGGCCGTGTATTACTGT 55 CDR3-5 AGGCCTCGGACACCGCCATGTATTACTGT 56DJ gene-back JH-U1 CTGAGGAGACGGTGACC 57 V-DJ fusion H48SfiI-forGCGATGGCCCAGCCGGCCATGGCCCAGRTGC 58 AGCTGGTRSAGTC H49SfiI-forGCGATGGCCCAGCCGGCCATGGCCCAGRTCA 59 CCTTGARGGAGTC H50SfiI-forGCGATGGCCCAGCCGGCCATGGCCCAGGTRC 60 AGCTRCAGSAGT H47SfiI-backGGAATTCGGCCCCCGAGGCCTGARGAGACRG 61 TGACC cf.) R: A or G; S: C or G

To obtain a VL gene, 1^(st) PCR was performed using each of primers(Table 2) for lambda gene amplification and primers (Table 3) for kappagene amplification, and each of the amplified fragments was subjected to2^(nd) PCR using primers (lambda: SEQ ID NOS. 76-81, and kappa: SEQ IDNOS. 106-108) having a BstXI digestion site at the 5′ end and the 3′end.

TABLE 2 Primer sequence for amplifying Lambda gene fragment SEQ ID NO:Vλ forward L01 CAGYCTGTGCTGACTCAG 62 L03 CAGCCTGTGCTGACTCAAT 63 L06TCCTATGAGCTGACWCAG 64 L15 CAGYCTGTGCTGACTCAGCCGT 65 L20CAGTCTGTGCTGACGCAGCCG 66 L23 CAGTCTGCCCTGACTCAGCCTC 67 L24CAGTCTGCCCTGACTCAGCCTG 68 L25 CAGRCTGTGGTGACYCAGGAGCCCTCAC 69 L26CAGRCTGTGGTGACYCAGGAGCCATCGT 70 L28 TCCTATGAGCTGACWCAGCCACT 71 L34BAATTTTATGCTGACTCAGCCC 72 Vλ back L35 CCTCCTCCACCTAGGACGGTGACCTTGG 73TCCCAGTT L36 CCTCCTCCACCTAGGACGGTCAGCTTGG 74 TCCCTCCG L37CCTCCTCCACCGAGGGCGGTCAGCTGGG 75 TGCCTCCT Vλ 2nd PCR (BstXI) L34BstXI-forGGTGGATCCAGCGGTGTGGGTTCCAATT 76 TTATGCTGACTCAGCCC L40BStXI-forGGTGGATCCAGCGGTGTGGGTTCCCAGY 77 CTGTGCTGACTCAGCC L41BstXI-forGGTGGATCCAGCGGTGTGGGTTCCCAGC 78 CTGTGCTGACTCAATC L42BstXI-forGGTGGATCCAGCGGTGTGGGTTCCCAGT 79 CTGCCCTGACTCAGCC L43BstXI-forGGTGGATCCAGCGGTGTGGGTTCCCAGR 80 CTGTGGTGACYCAGGA L44BstXI-forGGTGGATCCAGCGGTGTGGGTTCCTCCT 81 ATGAGCTGACWCAG L38BstXI-backGAATTCCACGAGGCTGGCTCCTCCACCK 82 AGGRCGGT cf) K: G or T; R: A or G; Y: Tor C; W: A or T

TABLE 3 Primer sequence for amplifying Kappa gene fragment SEQ ID NO: Vκforward K12 GACATCCAGATGACCCAGTCTCCATCCTCCC 83 K13GACATCCAGATGACCCAGTCTCCATCCTCA 84 K14 GACATCCAGATGACCCAGTCTCCATCTTCYG 85K15 GACATCCAGATGACCCAGTCTCCTTCCA 86 K16 AACATCCAGATGACCCAGTCTCCATCTGCCA87 K17 AACATCCAGATGACCCAGTCTCCATCCTT 88 K18 GCCATCCAGTTGACCCAGTCTCCAT 89K19 GCCATCCGGATGACCCAGTCTCCATTCTCC 90 K20 GTCATCTGGATGACCCAGTCTCCATCCTTA91 K21 GATATTGTGATGACCCAGACTCCACTCTCTCTGT 92 K22GATATTGTGATGACCCAGACTCCACTCTCCCTGC 93 K23GATATTGTGATGACCCAGACTCCACTCTCCTCA 94 K24 GATRTTGTGATGACTCAGTCTCCACTCTC95 K25 GAAATTGTGTTGACRCAGTCTCCAG 96 K27 GACATCGTGATGACCCAGTCTCCAG 97VKA1 GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCC 98 CGTCACCCTTGGAC VK10GAAATTGTGCTGACTCAGTCTCCAGACTTT 99 VK30GACATCCAGATGACCCAGTCTCCATCCTCCCTGTC 100 TGCATCTGTAGGAG Vκ back K28TCCTCCACGTTTGATTTCCACCTTGGTCCCTTG 101 K29TCCTCCACGTTTGATCTCCAGCTTGGTCCCC 102 K30 TCCTCCACGTTTGATATCCACTTTGGTCCCAG103 K31 TCCTCCACGTTTGATCTCCACCTTGGTCCCTCC 104 K32TCCTCCACGTTTAATCTCCAGTCGTGTCCCT 105 Vκ BstXI K33BstXI-forGGTGGATCCAGCGGTGTGGGTTCCGACATCCAGA 106 TGACCCAGTCTCC K34BstXI-forGGTGGATCCAGCGGTGTGGGTTCCGATATTGTGA 107 TGACCCAGWCTCC K36BstXI-GAATTCCACGAGGCTGGCTCCTCCACGTTTGATH 108 back TCCA cf) H: A, C or T; W: Aor T

To introduce the VH gene fragment and the VL gene fragment into aphagemid vector, a pAK100 vector (Krebber, A. et al., J. Immunol.Method., 201:35, 1997) was used. To introduce the VL gene fragment,three BstXI domains (236, 365 and 488) present in the lac repressor gene(lad) of the pAK100 vector were mutated using the Quikchangesite-specific mutagenesis kit (Stratagene, USA). Using the modifiedpAK100 vector, to prepare a backbone vector for the construction of theScFv library, the heavy chain V gene, amplified using the H05 primer(SEQ ID NO: 29) and the H230 primer (SEQ ID NO: 49), and the DJ genefragment, amplified using the CDR3-3 primer (SEQ ID NO: 54) and theJH-U1 primer (SEQ ID NO: 57), were subjected to 2^(nd) PCR with H48SfiI(SEQ ID NO: 58)/H47SfiI (SEQ ID NO: 61), and the resulting heavy chainvariable region was digested with SfiI and ligated into the modifiedpAK100 vector digested with the same enzyme. To introduce a light chainand a linker, the heavy chain region was amplified using primers(forward: SEQ ID NO: 109; and backward: SEQ ID NO: 110), and the lightchain sequence of the human 4-1BB antibody (LB506) (Korean PatentPublication 2000-0034847) was amplified using each of primers (forward:SEQ ID NO: 111; and backward: SEQ ID NO: 112). The amplified fragmentswere inserted into the modified pAK100 vector having the heavy chainvariable domain introduced therein, using XbaI/EcoRI, thus preparing anantibody library backbone vector.

SEQ ID NO: 109: 5′-CGAATTTCTAGATAACGA-3′ SEQ ID NO: 110:5′-CCTCCGCCACTACCTCCTCCTCCGAGGCCCCCGAGGCCTGA-3′ SEQ ID NO: 111:5′-GGTAGTGGCGGAGGAGGCTCCGGTGGA TCCAGCGGTGTGG GTTCCGATATTGTG-3′SEQ ID NO: 112: 5′-CTCGAATTCCCACGAGGCTGGCTCCTCCACGTTTGATTTC-3′

In order to introduce light chain variable regions, each of amplifiedlight chain (κ, λ) variable regions was digested with BstXI and insertedinto the antibody library backbone vector. The resulting plasmid wasdigested with a SfiI restriction enzyme and ligated with a heavy chainvariable region-amplified PCR fragment previously digested with SfiI.The ligated plasmid was transfected into ElectroTen-Blue competent cells(Stratagene, USA). As a result, a ScFv phage library having a diversityof about 10¹¹ was collected from the colony.

Example 3 Biopanning

The library stock constructed in Example 2 was grown to the log phaseand rescued with the M13K07 helper phage (GE healthcare, USA). Theresulting library was amplified in 2xYT medium (2xYT/C/K; containing 34μg/ml of chloramphenicol and 70 μg/ml of kanamycin and supplemented with1 mM IPTG) at 30° C. overnight.

Phage stock was precipitated in 20% PEG6000/2.5M NaCl and resuspended inPBS. Resuspended phage stock was incubated in 2% skimmed milk/PBSsolution containing 500 μg/ml of a human Fc protein at 37° C. for 1 hourin order to remove phages showing anti-human Fc.

The KDR (human VEGFR-2) used as an antigen was KDR(ECD1-3)-Fc comprisingIgG-like domains 1, 2 and 3 of the extracellular domain of KDR. TheKDR(ECD1-3)-Fc stable cell line prepared in Example 1 was cultured, andKDR(ECD1-3)-Fc was purified from the cultured cell line.

Maxisorb Star tubes (Nunc, Denmark) coated with KDR(ECD1-3)-Fc (10μg/ml) were first blocked with 2% skimmed milk/PBS at room temperaturefor 2 hours, and then inoculated with 5.4×10¹² pfu of the phage stock atroom temperature for 1 hour.

The tubes were washed 10 times with PBST (PBS containing 0.1% Tween 20),and then washed 10 times with PBS. The bound phage was eluted with 1 mlof 100 mM fresh triethylamine solution at room temperature for 10minutes. The eluted phage was left to stand together with 10 ml ofmid-log-phase XL1-Blue cells at 37° C. for 30 minutes, and thenshake-cultured for 30 minutes. Then, the infected XL1-Blue cells werecultured in a 1% glucose-containing 2×YT/C plate at 30° C. overnight.Following the first panning, the second and third panning processes wereperformed by coating KDR(ECD1-3)-Fc into a 96-well plate (Nunc, USA)instead of the maxisorp tube. After the third panning was performed, theKDR neutralizing ability of the obtained phage was analyzed through VEGFcompetition assays.

For VEGF competition assays, a microplate coated with 200 ng of VEGF165(R&D system) overnight was allowed to react with 2% skimmed milk/PBS at37° C. for 2 hours. The microplate was washed with PBS, and then amixture, obtained by reacting 10 ng of KDR (ECD1-3)-Fc with variousamounts of phage at room temperature for 1 hour, was placed in each wellof the plate and allowed to react at room temperature for 2 hours. Thereaction solution was washed with PBS, allowed to react with a rabbitanti-KDR antibody (Reliatech, Germany) at 37° C. for 1 hour, and allowedto react with an HRP (horse radish peroxidase)-conjugated goatanti-rabbit antibody (Abcam, UK) at 37° C. for 1 hour. After thecompletion of the reaction, each well was color-developed with a TMBsolution (Sigma), and then measured for absorbance at 450 nm (FIG. 4).

As a result, it was seen that 6A6, 6H1, 6G1 and 6C1 could all inhibitthe binding of VEGF to KDR, and among them, 6A6 and 6H1 showed thehighest ability to neutralize VEGF. Also, 6A6 and 6H1 were shown to havea binding affinity similar to that of a reconstructed 1C11 (hereinafterreferred to as 1C11) phage obtained in Example 4. The DNA sequences,amino acid sequences and CDR sequences of 6A6 (TTAC-0001) ScFv are shownin FIG. 5.

Also, the base sequences and amino acid sequences of 6A6 (TTAC-0001)ScFv were expressed as heavy chain CDR 1 (SEQ ID NO: 113 and SEQ ID NO:114), heavy chain CDR 2 (SEQ ID NO: 115 and SEQ ID NO: 116), heavy chainCDR 3 (SEQ ID NO: 117 and SEQ ID NO: 118), light chain CDR 1 (SEQ ID NO:119 and SEQ ID NO: 120), light chain CDR 2 (SEQ ID NO: 121 and SEQ IDNO: 122), light chain CDR 3 (SEQ ID NO: 123 and SEQ ID NO: 124), heavychain variable regions (SEQ ID NO: 125 and SEQ ID NO: 20), light chainvariable regions (SEQ ID NO: 126 and SEQ ID NO: 1), IgG heavy chainregions (SEQ ID NO: 127 and SEQ ID NO: 128), and IgG light chain regions(SEQ ID NO: 129 and SEQ ID NO: 130).

Example 4 Construction of Reconstructed IMC-1121(rIMC-1121) andIMC-1C11(rIMC-1C11) Phage Vectors

In order to obtain IMC-1C11 ScFv (PCT/US2001/10504) and IMC-1121 ScFv(PCT/US2002/006762) phage particles (Imclone) to be used as positivecontrol groups, the ScFv region of each antibody was cloned into the pAKvector.

For IMC-1C11, a light chain variable gene was cloned using, as atemplate, a pTA-d9-07 clone (LG Life Sciences) obtained from a mousenaΔve antibody library (LG Life Sciences). The clone was amplified byPCR using the LR and LF primers shown in Table 4, and the amplifiedlight chain variable gene was digested with BstXI and ligated into alibrary backbone vector pretreated with BstXI. A heavy chain variablegene was amplified by PCR using a pTA-A5N2-10 clone (LG Life Sciences)as a template with the primers shown in Table 4. After each of the PCRreactions was performed using each of the primer pairs HF1-RI(A),HF2-HR2(B), HF3-HR3(C) and HF4-HR4(D), and each of the amplifiedfragments was amplified by overlap PCR using A-B (HF1-HR2 primer set)and C-D (HF3-HR4 primer pair), and then amplified by overlap PCR usingA-B-C-D (HF1-HR4 primer pair). Then, each of the amplified fragments wastreated with SfiI and ligated with the 1C11 light chain gene-containinglibrary backbone vector treated with SfiI. Table 4 shows the PelB signalsequence and DNA sequence to amber (TGA) codon of the phage vector(pAK-r1c11).

TABLE 4 LR and LF primer SEQ ID Name Sequence NO: pTA-d9_07GACATTGTTC TCATCCAGTC TCCAGCAATC ATGTCTGCAT 131 light chainCTCCAGGGGA GAAGGTCACC ATAACCTGCA GTGCCAGCTCAAGTGTAAGT TACATGCACT GGTTCCAGCA GAAGCCAGGCACTTCTCCCA AACTCTGGAT TTATAGCACA TCCAACCTGGCTTCTGGAGT CCCTGCTCGC TTCAGTGGCA GTGGATCTGGGACCTCTTAC TCTCTCACAA TCAGCCGAAT GGAGGCTGAAGATGCTGCCA CTTATTACTG CCAGCAAAGG AGTAGTTACCCATTCACGTT CGGCTCGGGG ACAAAGTTGG AAATAAAA pTA-A5N²-10CAGGTTCAGC TCCAGCAGTC TGGGGCAGAG CTTGTGAGGT 132 heavy chainCAGGGGCCTC AGTCAAGTTG TCCTGCACAG CTTCTGGCTTCAACATTAAA GACTACTATA TGCACTGGGT GAAGCAGAGGCCTGAACAGG GCCTGGAGTG GATTGGATGG ATTGATCCTGCGAATGGTAA TACTAAATATGACCCGAAGT TCCAGGGCAAGGCCACTATA ACAGCAGACA CATCCTCCAA CACAGCCTACCTGCAGCTCA GCAGCCTGAC ATCTGAGGAC ACTGCCGTCTATTACTGTGC TAGATGGGAC TGGTACTTCG ATGTCTGGGG CGCAGGGACC ACGGTCACCG TTTCCLF CTGCAGAACC AGCGGTGTGG GTTCCGACAT CGAGCTCACT 133 CAGTCTCCAT G LRCTGCAGAACC ACGAGGCTGG CTCCTCCACG TTTTATTTCC 134 AGCTTGGTCC CCG HF1CGGCCCAGCC GGCCATGGCC CAGGTCAAGC TGCAGCAGTC 135TGGGGCAGAG CTTGTGGGGT CAGGGGCC HF2 GGCTTCAACA TTAAAGACTT CTATATGCA 136HF3 GATTATGCCC CGAAGTTCCA GGGCAAGGCC ACCATGACTG 137 CAGACTCATC CTCCA HF4TACTGTAATG CATACTATGG TGACTACGAA GGCTACTGGG 138 GCCAA HR1GTCTTTAATG TTGAAGCCAG AAGTTGTGCA G 139 HR2ACTTCGGGGC ATAATCAGAA TCACCATTCT CAGGATCAAT 140 CCATCCAATC HR3GTATGCATTA CAGTAATAG 141 HR4 CCGAGGCCCC CGAGGCCTGA GGAGACGGTG ACCGTGGTCC142 CTTGGCCCCA GTAGCCTTCG TA r1C11-ScFvATGAAATACC TATTGCCTAC GGCAGCCGCTGGATTGTTAT 143 DNATACTCGCGGC CCAGCCGGCC ATGGCCCAGG TCAAGCTGCAGCAGTCTGGG GCAGAGCTTG TGGGGTCAGG GGCCTCAGTCAAATTGTCCT GCACAACTTC TGGCTTCAAC ATTAAAGACTTCTATATGCA CTGGGTGAAG CAGAGGCCTG AACAGGGCCTGGAGTGGATT GGATGGATTG TCCTGAGAA TGGTGATTCTGATTATGCCC CGAAGTTCCA GGGCAAGGCC ACCATGACTGCAGACTCATC CTCCAACACA GCCTACCTGC AGCTCAGCAGCCTGACATCT GAGGACACTG CCGTCTATTA CTGTAATGCATACTATGGTG ACTACGAAGG CTACTGGGGC CAAGGGACCACGGTCACCGT CTCCTCAGGC CTCGGGGGCC TCGGAGGAGGAGGTAGTGGC GGAGGAGGCT CCGGTGGATC CAGCGGTGTGGGTTCCGACA TCGAGCTCAC TCAGTCTCCA GCAATCATGTCTGCATCTCC AGGGGAGAAG GTCACCATAA CCTGCAGTGCCAGCTCAAGT GTAAGTTACA TGCACTGGTT CCAGCAGAAGCCAGGCACTT CTCCCAAACT CTGGATTTAT AGCACATCCAACCTGGATTA TGGAGTCCCT GCTCGCTTCA GTGGCAGTGGATCTGGGACC TCTTACTCTC TCACAATCAG CCGAATGGAGGCTGAAGATG CTGCCACTTA TTACTGCCAG CAAAGGAGTAGTTACCCATT CACGTTCGGC TCGGGGACCA AGCTGGAAATAAAACGTGGA GGAGCCAGCC TCGTGGAATT CGAGCAGAAG CTGATCTCTG AGGAAGACCT GTAG

In order to obtain IMC-1121, 6G1 was used as a template to clone a lightchain variable region. The 6G1 template was amplified by PCR using theprimer pairs LF-KR1(A), LF1-LR2(B), LF2-LR3(C) and LF3-LR4(D), wasamplified by overlap PCR using A-B (LF-LR2 primer set) and C-D (LF2-LRprimer pair), and was then amplified by overlap PCR using A-B-C-D (LF-LRprimer pair). The obtained PCR fragment was treated with BstXI andinserted into the library backbone vector (reconstructed IMC-1121;hereinafter referred to as IMC-1121). The primers used herein are shownin Table 5.

For the heavy chain variable region, the YGKL-136 clone having asequence closest to IMC-1121 among the clone sequences obtained from thehuman naΔve scFv library (Example 2) was used as a template. TheYGKL-136 clone was amplified by PCR using each of the primer pairsHF-HR1(A), HF1-HR2(B) and HF2-HR(C), was subjected to A+B overlapPCR(HF-HR2 primer pair), and was then subjected to A+B and C overlapPCR(HF-HR primer pair). The produced PCR fragment was treated with SfiIand ligated into a light chain-containing library backbone vector.

TABLE 5 LF and HF primer for cloning light chain variable region SEQ IDName Sequence NO: YGKL-136 GAGGTGCAGC TGGTGGAGTC TGGGGGAGGC CTGGTCAAGC144 heavy chain CTGGGGGGTC CCTGAGACTC TCCTGTGCAG CCTCTGGATT (template)CACCTTCAGT AGCTATAGCA TGAACTGGGT CCGCCAGGCTCCAGGGAAGG GGCTGGAGTG GGTCTCATCC ATTAGTAGTAGTAGTAGTTA CATACACTAC GCAGACTCAG TGAAGGGCCGATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTATCTGCAAATGA ACAGTCTGAG AGCCGAGGAC ACGGCCGTGTATTACTGTGC GAGAGTCACA GATGCTTTTG ATATCTGGGGCCCCGGAACC CTGGTCACCG TCTCCTCA 6G1 lightGACATCCAGA TGACCCAGTC TCCATCTTCC GTGTCTGCAT 145 chainCTGTAGGAGA CAGAGTCACC ATCACTTGTC GGGCGAGTCA (template)GGGTATTAGC AGCTATTTAG GCTGGTATCA GCAGAAACCAGGGAAAGCCC CTAAGCTCCT GATCTATGCT GCATCCAATTTGCAAACAGG GGTCCCGCCA AGGTTCAGCG GCAGTGGATCCHHHACAAGT TTCACTCTCA CCCTCAATAA TGTGCAGCCTGAAGATTCTG CAACTTACTA TTGTCAACAG GCTGACAGTTTCCCTCTTTC GGCGGAGGGA CCAAAGTGGA AATCAAACGT GAGGAGCC LF primerCCCCAGCGGT GTGGGTTCCG ACA 146 LR1 primerTGGTGACTCT GTCTCCTATA GATGCAGACA CGGATGAT 147 LF1 primerTCTATAGGAG ACAGAGTCAC CA 148 LR2 primerTACCAGCCTA ACCAGTTGTC AATACCCTGA CTCGCCCG 149 LF2 primerTTGACAACTG GTTAGGCTGG TATCAGCAGA AACCAGGG 150 AAA LR3 primerACCTTGATGG GACCCCTGTG TCCAAATTGG ATGCATCATA 151 GATCAGGAGC TT LR primerCCCCACGAGG CTGGCTCCTC CA 152 HF primerCCGGCCCAGC CGGCCATGGC CGAGGTGCAG CTGGTGCAGT 153 CTGGGGGAGG CCTGGTCAHF1 primer GTAGTAGTAG TAGTTACATA TACTACGCAG ACTCAGTGA 154 HF2 primerTTACTGTGCG AGAGTCACAG ATGCTTTTGA TATCTGGGGC 155 CAAGGGACAA HR1 primerTCACTGAGTC TGCGTAGTAT ATGTAACTAC TACTACT 156 HR2 primerCTGTGACTCT CGCACAGTAA TACA 157 HR primerCCGGCCCCCG AGGCCTGAGG AGACGGTGAC CATTGTCCCT 158 TGGCCCCAG r1121-ScFvATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT 159 DNATACTCGCGGC CCAGCCGGCC ATGGCCGAGG TGCAGCTGGTGCAGTCTGGG GGAGGCCTGG TCAAGCCTGG GGGGTCCCTGAGACTCTCCT GTGCAGCCTC TGGATTCACC TTCAGTAGCTATAGCATGAA CTGGGTCCGC CAGGCTCCAG GGAAGGGGCTGGAGTGGGTC TCATCCATTA GTAGTAGTAG TAGTTACATATACTACGCAG ACTCAGTGAA GGGCCGATTC ACCATCTCCAGAGACAACGC CAAGAACTCA CTGTATCTGC AAATGAACAGTCTGAGAGCC GAGGACACGG CCGTGTATTA CTGTGCGAGAGTCACAGATG CTTTTGATAT CTGGGGCCAA GGGACAATGGTCACCGTCTC CTCAGGCCTC GGGGGCCTCG GAGGAGGAGGTAGTGGCGGA GGAGGCTCCG GTGGATCCAG CGGTGTGGGTTCCGACATCC AGATGACCCA GTCTCCATCT TCCGTGTCTGCATCTATAGG AGACAGAGTC ACCATCACTT GTCGGGCGAGTCAGGGTATT GACAACTGGT TAGGCTGGTA TCAGCAGAAACCTGGGAAAG CCCCTAAACT CCTGATCTAC GATGCATCCAATTTGGACAC AGGGGTCCCA TCAAGGTTCA GTGGAAGTGGATCTGGGACA TATTTTACTC TCACCATCAG TAGCCTGCAAGCTGAAGATT TTGCAGTTTA TTTCTGTCAA CAGGCTAAAGCTTTTCCTCC CACTTTCGGC GGAGGGACCA AGGTGGACATCAAACGTGGA GGAGCCAGCC TCGTGGAATT CGAGCAGAAG CTGATCTCTG AGGAAGACCT GTGA

Example 5 Production and Purification of Soluble ScFv

To prepare soluble 6A6 ScFv, pAK-6A6, having a pelB sequence and an ScFvsequence, was digested with EcoRI and XbaI to obtain a fragment, havingthe pelB sequence and the ScFv sequence. The fragment was inserted intothe pET21b vector (Novagen, USA) using the same restriction enzymes. Toadd an myc tag to the vector inserted with the fragment having the pelBsequence and the ScFv sequence, the pET21b vector, inserted with thepelB sequence and the ScFv sequence, as a template, was amplified by PCRusing primers (mycFor: SEQ ID NO: 160, and mycRev: SEQ ID NO: 161), andthe PCR fragment was ligatged into the vector having the pelB sequenceand the ScFv sequence, using EcoRI and XhoI, thus constructingpET21b-KDR 6A6.

SEQ ID NO: 160: 5′-GAGCCAGCCTCGTGGAATTCGAACAAAAA-3′ SEQ ID NO: 161:5′-TGCTCGAGAT TCAGATCCTC TTCTGAGATG AGTTTTTGTT GAATTCCACG AGGCT-3′

For kinetic measurement, a V5 tag sequence (GKPIPNPLLGLDST) was insertedin an XhoI site downstream of the myc tag in the following manner. Theresulting sequence was amplified by PCR using primers (V5-For: SEQ IDNO: 162, and V5-Rev: SEQ ID NO: 163) amplifying the EcoR1 and V5 tagsequence-containing XhoI digestion sites of the pET21b-KDR 6A6, and theamplified frasgment was digested with EcoRI and XhoI and ligated intothe pET21b-KDR 6A6 digested with the same restriction enzymes, thusconstructing pETV-KDR 6A6. The constructed pETV-KDR 6A6 was transformedinto E. coli BL21(DE3).

SEQ ID NO: 162: 5′-CCAGCCTCGTGGAATTC GAAC-3′ SEQ ID NO: 163:5′-CCGCTCGAG GGTGGAGTC CAGACCTAATAG AGGGTTTGGGATCGG CTTTCCATTCAGATC CTCTTCTGA-3′

The E. coli BL21(DE3) cells transformed with the pETV-KDR 6A6 werecultured to express the soluble ScFv protein and centrifuged, and aperiplasmic fraction was collected from the cells using 50 mM Tris (pH8.0) solution containing 20% sucrose and 200 μg/ml of a lysozyme andprotease inhibitor cocktail (Roche, Swiss). The obtained fraction waspurified using Ni-NTI affinity chromatography (Hisprep, GE healthcare,USA) and ion exchange chromatography (Q-sepharose, SP-sepharose, GEhealthcare, USA), thus obtaining an ScFv protein.

For 6A6, the Hisprep column was equilibrated using a solution containing20 mM imidazole, 0.4M NaCl and 1×PBS, and the periplasmic fraction wasplaced in the column and eluted with 300 mM imidazole-containingsolution (300 mM imidazole, 0.4M NaCl/1×PBS). The eluted protein wasdialyzed with 50 mM imidazole (pH 6.7) solution, and then eluted usingcation exchange chromatography while increasing the concentration ofNaCl to 0.5M. The eluted protein was concentrated (centriprep YM10,milipore, USA), and then dialyzed and stored in PBS solution. FIG. 6shows the results of SDS-PAGE of the purified 6A6 ScFv.

Example 6 VEGF Competition Assays with VEGF

In order to examine whether the isolated ScFv can inhibit the binding ofKDR to VEGF, competition assays were performed. For this purpose, 20 ngof VEGF165 was coated into a 96-well microtiter plate at roomtemperature overnight, and then allowed to react with 2% skimmedmilk/PBS at 37° C. for 2 hours. After the completion of the reaction,the plate was washed with PBS, and then mixture solutions, obtained byallowing 100 ng of Fc-digested KDR(ECD1-3) to react with various amountsof ScFv at room temperature for 1 hour, were placed in the microtiterplate and allowed to react at room temperature for 2 hours. After thecompletion of the reaction, the plate was washed with PBS, and then ananti-KDR mouse antibody (5 μg/ml, Reliatech, Germany) was added theretoand allowed to react at 37° C. for 1 hour. Then, a 1:5000 dilution of anHRP-conjugated goat anti-mouse antibody (Abcam, UK) was added theretoand allowed to react for 1 hours, and then TMB solution was addedthereto and allowed to react. Then, the cells in each well of the platewere measured for absorbance at 450 nm and 650 nm. FIG. 7 shows theresults of VEGF competitive assays with the anti-KDR-ScFv purified inExample 5. As shown in FIG. 7, it can be seen that only 6A6-ScFv shows apotent ability to neutralize KDR.

Example 7 Epitope Mapping with Anti-KDR ScFv Antibody

In order to examine which anti-KDR ScFv antibodies bind to which domainof extracellular domains 1-3 of KDR, 3 μg/ml of each of the KDR(ECD1-2),KDR(ECD2-3) and KDR(ECD 1-3)-Fc prepared according to the method ofExample 1 was coated into a 96-well plate by reaction at 37° C. for 2hours. After the completion of the reaction, the plate was washed withPBS, and then the portion of the plate, which has not been coated withthe KDR protein, was blocked with 2% skimmed milk/PBS. Then, the platewas washed again with PBS, and then 330 nM anti-KDR ScFv antibody wasadded thereto and allowed to react at 37° C. for 1 hour and 30 minutes.After the completion of the reaction, the plate was washed again withPBS, and then a 1:500 dilution of an HRP-conjugated rabbit anti-6×Hisantibody (Abcam, UK) was added thereto and allowed to react at 37° C.for 1 hour. Then, the cells in each well were color-developed with TMBsolution and measured for absorbance at 450 nm.

As a result, it could be seen that 6A6 was bound to the extracellulardomain 3 of KDR in the same manner as IMC-1121 (FIG. 8). However, 6G1and 6C1 were more strongly bound to the domain 1, even though theabsorbance was low.

Example 8 Expression and Purification of IgG

For expression in the form of whole IgG, heavy chain and light chainexpression vectors, each comprising a whole constant region, wereprepared. For the heavy chain expression vector, a pIgGHD vector(Aprogen, Korea) having a heavy chain backbone of human 4-1 bb wastreated with SfiI, and then ligated with a fragment, obtained bytreating the heavy chain variable region of pAK-ScFv with SfiI, thusconstructing an expression vector pIgGHD-6A6Hvy, comprising a wholeconstant region and a heavy chain region (FIG. 9).

For the light chain expression vector, a pIgGLD vector (Aprogen, Korea)having a light chain backbone of human 4-1 bb was treated with BstXI,and then ligated with a fragment, obtained by treating the light chainvariable region of pAK-ScFv with BstXI, thus constructing an expressionvector pIgGLD-6A6Lgt, comprising a whole constant region and a lightchain region (FIG. 10). In the case of IMC-1C11 and 1121, IgG expressionvectors were constructed in the same manner as described above.

For the expression of IgG, the same amount of the light chain expressionvector (pIgGHD-6A6Hvy for the 6A6 clone) and the heavy chain expressionvector (pIgGLD-6A6Lgt for the 6A6 clone) were co-transfected into CHODG44 cells (Aprogen, Korea). The co-transfected cells were cultured inα-MEM medium containing 10% dFBS and 500 μg/ml of G418, and then a clonehaving the highest protein expression level was selected, while MTX wasadded thereto at a concentration ranging from 10 nM to 700 nM.

For the expression of antibodies, the cells were cultured in CHO-SF2medium, containing 700 nM MTX, at 37° C., and the culture was collected.6A6-IgG was purified from the combined supernatant by affinitychromatography using a protein A column (GE healthcare, USA) accordingto the manufacturer's protocol. The supernatant was poured into theprotein A column equilibrated with a solution containing 20 mM sodiumphosphate (pH 7.0) and 100 mM NaCl and was washed with a solutioncontaining 20 mM sodium phosphate (pH 7.0), 1 mM EDTA and 500 mM NaCl.Then, the protein was eluted with a 0.1 M glycine-HCl (pH 3.3) solutioncontaining 100 mM NaCl. The eluted protein was neutralized with 1 MTris. The eluted protein was mixed with 5 mM sodium phosphate buffer (pH6.0) at a ratio of 1:1, and then poured into a prepacked SP-Sepharosecolumn (GE healthcare) equilibrated with 5 mM sodium phosphate (pH 6.0)containing 50 mM NaCl. The protein bound to the column was eluted with asodium phosphate buffer (pH 7.0) containing 50 mM NaCl and was pouredinto a prepacked Q-sepharose column (GE healthcare) equilibrated with anelution buffer, and unbound protein was collected. The collected proteinwas concentrated with 30 Kd vivaspin 20 (Sartorius) and dialyzed withPBS. FIG. 11 shows the results of SDS-PAGE of the 6A6 IgG proteinpurified according to the above-described method.

Example 9 Competition Assays of Anti-KDR IgG with Various VEGFs

Competition assays of anti-KDR IgG with VEGF were carried out in thesame manner as the VEGF competition assays of KDR ScFv, conducted inExample 6 using VEGF165. As a result, 6A6 IgG among anti-IgGs showed thehighest ability to neutralize KDR, similar to the results of thecompetition assays conducted with ScFv, and it showed KDR neutralizingability similar to that of the IMC-1121 anti-KDR IgG reconstructed onthe basis of the amino acid sequence (FIG. 12).

Also, in order to examine the binding and competition of 6A6 IgG withisotypes and VEGF families other than VEGF165, 200 ng of each ofVEGF121, VEGF165, VEGF-C, VEGF-D and VEGF-E was coated into a 96-wellplate, and then competition assays were carried out in the same manneras in Example 6. As a result, 6A6 IgG showed VEGF neutralizing abilityby binding to VEGF121, VEGF165 and VEGF-E, which belong to VEGF-A, andit did not bind to VEGF-C and VEGF-D (FIG. 13).

Example 10 Analysis of Binding Affinity of Anti-KDR ScFv and IgG

The binding affinity of the antibodies to KDR (VEGFR-2) was measuredwith BIAcore (GE healthcare). In the case of ScFv, KDR(ECD1-3)-Fc wasimmobilized onto a CM5 chip (GE healthcare, Sweden) according to themanufacturer's manual, and in the case of V5-tagged ScFv, a V5 antibody(Abchem, UK) was immobilized onto the chip. The V5-tagged ScFv was boundto the CM5 chip having the V5 antibody immobilized thereon, and thenFc-free KDR(ECD1-3) was allowed to run on the chip surface, thusobtaining sensorgrams. In the case of IgG, as in the case of the ScFvhaving no V5, KDR(ECD1-3)-Fc was immobilized onto the CM5 chip, and thenvarious amounts of the antibody was allowed to run on the chip surface,thus obtaining sensorgrams. Based on the sensorgram obtained at eachconcentration, the kinetic constants k_(on) and k_(off) were measured,and K_(d) was calculated from the ratio of the kinetic constantsk_(off)/k_(on) (Table 6).

As a result, it was confirmed that, among various ScFvs, ScFv having ahigh binding affinity for KDR was 6A6. Also, when 6A6 was converted inthe form of IgG, the Kd value thereof was about 2-fold lower than thatof IMC-1121. This suggests that 6A6 IgG was more strongly bound to KDRcompared to IMC-1121.

TABLE 6 K_(d) (M) value of anti-KDR ScFv, IgG IgG ScFv ScFv-V5 k_(on)(1/Ms) k_(off) (1/s) K_(d) (M) 6A6 1.11E−08 6.93E−09 3.17E+05 7.3E−05 2.3E−10  6H1 N/A N/A 5.02E+04 7.20E−03 1.43E−07 6G1 4.11E−07 3.31E−089.06E+04 5.48E−03 6.05E−08 6C1 4.31E−08 N/A 1.38E+05 9.58E−03 6.95E−08IMC-1121b N/A N/A 2.27E+05 8.75E−05 3.85E−10 * N/A (not applicable)

Example 11 Analysis of KDR Neutralizing Ability of Anti-KDR-IgG in HUVECCells Using FACS Analysis

Primary-cultured HUVEC cells were cultured in serum-free mediumovernight to induce the overexpression of KDR, and then the cells wereharvested, washed three times with PBS. The washed cells were allowed toreact with 6A6 or IMC-1C11 IgG (10 μg/ml) at 4° C. for 1 hour, and thenallowed to react with an FITC-labeled rabbit anti-human IgG antibody(Abchem, UK) for 60 minutes. After completion of the reaction, the cellswere washed and analyzed with a flow cytometer (FACS; model EPICS9,Coulter Corp., USA).

As a result, as shown in FIG. 14, IMC-1C11 and 6A6 recognized the KDR ofHUVEC cells at the same level.

Also, in order to examine competitive inhibitory ability against VEGF,HUVEC cells were cultured in a serum-free condition overnight to inducethe expression of KDR, and then the cells were harvested and washedthree times with PBS. The washed cells were allowed to react with 20ng/ml of VEGF at room temperature for 30 minutes. After the completionof the reaction, the cells were allowed to react with 6A6-IgG andIMC-1C11 IgG at 4° C. for 1 hour, and then allowed to react with anFITC-labeled rabbit anti-human IgG antibody at 4° C. for 30 minutes.

As a result, as shown in FIG. 15, it was observed that the twoantibodies all showed a signal of binding to VEGF165, indicating that6A6-IgG was competitively bound to VEGF. Also, in the VEGF competitionassays, 6A6-IgG and IMC-1C11 IgG showed the same level ofKDR-neutralizing ability, but the KDR neutralizing ability in actualliving cells was about two-fold higher for 6A6-IgG than for IMC-1C11IgG.

Example 12 Analysis of KDR Neutralizing Ability of Anti KDR-IgG in K562Cells

In order to examine the KDR binding affinity of the antibodies in KDRexpressing cells lines other than HUVEC cells, the expression of KDR inthe leukemia cell line K562 (ATCC CCL-243) was analyzed. FIG. 16 showsthe results of Western blot analysis for the expression of KDR in K562(ATCC CCL-243) cells. As shown in FIG. 16, K562 and HUVEC cellsexpressed KDR regardless of the presence or absence of serum. Thus, theK562 cells (ATCC CCL-243) were treated in the same manner as the HUVECcells of Example 11 and analyzed by FACS in order to examine whetherKDR-IgG could bind to the K562 cells.

As a result, as shown in FIG. 17, only 6A6-IgG was bound to the K562cells at a significant level, unlike the HUVEC cells. The results ofFACS assays through VEGF competition are shown in FIG. 18. As shown inFIG. 18, the K562 cells did not show a great change in the rate ofpositive cells, unlike the HUVEC cells. Although the reason is unclear,this is thought to be because the 6A6 antibody strongly binds to KDRexpressed on the surface of the K562 cells or regulates the growth ofthe cells using the autocrine loop mechanism of VEGF/KDR(VEGFR-2), andthus if VEGF is externally treated, the expression of KDR in the K562cells is induced, so that an increased amount of the KDR protein isexpressed on the cell surface, and the 6A6-IgG signal is increasedcompared to before the VEGF is externally treated. Also, as shown inFIG. 19, it was seen that 6A6-IgG could bind the KDR ofgleevec-resistant K562 cells (The Catholic University of Korea).

Example 13 Analysis of Inhibition of HUVEC Cell Proliferation by 6A6Antibody

The inhibition of HUVEC cell proliferation by anti-KDR-IgG was analyzedusing WST-1 reagent (Roche, Swiss). HUVEC cells were dispensed into eachwell of a gelatin-coated, 24-well culture plate at a concentration of2×10⁴ cells/well and cultured for 18 hours. Then, the cells were furthercultured in serum-free M199 medium (Sigma-aldrich, USA) for 4 hours, andthen 20 ng/ml of VEGF and various concentrations of 6A6 were addedthereto. Then, the WST-1 reagent was added thereto according to themanufacturer's manual, and after 1 hour and 4 hours, the cells weremeasured for absorbance at 450 nm and 690 nm (FIG. 20).

As a result, when the HUVEC cells were treated with VEGF, theproliferation thereof was increased by about three times, but when the6A6 antibody was added to the HUVEC cells, the proliferation of thecells was reduced in a concentration-dependent manner.

Example 14 Analysis of Effect of 6A6 Antibody on Inhibition of KDR andERK Phosphorylation

Sufficiently grown HUVEC cells were cultured in 1% FBS-containing M199medium for 6 hours, and then treated with VEGF and 6A6, IMC-1121 and 6C1antibodies at various concentrations for 10 minutes. Then, the cellswere lysed with 1 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA,137 mM NaCl, 1 mM Na₃VO₄, 1 mM PMSF, 10% glycerol, 1% Triton X-100) andcentrifuged, and the supernatant was treated with 1 μg/ml of ananti-KDR/Flk-1 antibody (Santa cruz Biotechnology, USA) at 4° C. for 3hours. The treated supernatant was incubated on protein-A agarose beads(Sigma-aldrich, USA) for 1 hour, and the immunoprecipitated protein waselectrophoresed on SDS-PAGE, and then analyzed by Western blot (FIG.21A).

As a result, it was observed that, when the cells were treated withVEGF, the phosphorylation of KDR was increased as expected, but thecells were treated with 6A6 or IMC-1121, the phosphorylation of KDR byVEGF was inhibited. Also, the 6C1 antibody had no effect on theneutralization of VEGF.

According to the above-described method, a test was carried out toexamine whether the phosphorylation of the kinase ERK known to receivethe signal of KDR would be inhibited. As a result, it was confirmed that6A6 and IMC-1121 inhibited the phosphorylation of ERK, but 6C1 did notsubstantially inhibit the phosphorylation of ERK as expected (FIG. 21B).

Example 15 Analysis of Inhibitory Effect of Anti-KDR-6A6 IgG on theChemotaxis of Endothelial Cells Induced by VEGF

In order to examine the inhibitory effect of 6A6-IgG on the migration ofHUVEC cells induced by VEGF, a transwell (Corning costar, USA) having a6.5-mm-diameter polycarbonate filter (8 μM pore size) was used. Thesurface of the lower layer of the filter was coated with 10 μg ofgelatin, and fresh M199 medium (containing 1% FBS) and VEGF were placedin the lower layer well of the filter. HUVEC cells were diluted in M199medium (containing 1% FBS) at a concentration of 1×10⁶/ml, variousconcentrations of the anti-KDR antibodies were added thereto and allowedto react at room temperature for 30 minutes. 100 μl of the reactionsolution was placed in the upper layer well and allowed to react at 37°C. for 4 hours. Then, the cells were stained with hematoxylin and eosin.Non-migrated cells were removed with cotton, and cells migrated into thelower layer well were observed with a microscope to measure the numberof the migrated cells. As a result, it was observed that the migrationof HUVEC cells induced by VEGF was inhibited by 6A6 in aconcentration-dependent manner (FIG. 22).

Example 16 Analysis of Inhibitory Effect of Anti-KDR-6A6 IgG onEndothelial Cell Tube Formation Induced by VEGF

In order to examine whether the 6A6 antibody inhibits HUVEC tubeformation induced by VEGF, 250 μl of growth factor-reduced matrigel(Collaborative biomedical products, USA) was placed in each well of a16-mm-diameter tissue culture plate and polymerized at 37° C. for 30minutes. HUVEC cells were suspended in M199 medium (containing 1% FBS),and various amounts of the antibody was mixed and allowed to react withthe cells. After 30 minutes, the cells were plated on the matrigel at aconcentration of 2×10⁵ cells/well, 10 ng/ml of VEGF was added thereto,and the cells were cultured for 20 hours. The cultured cells wereobserved with a microscope and imaged with an Image-Pro plus (Mediacybernetics, USA). As a result, it was observed that HUVEC tubeformation induced by VEGF was inhibited by 6A6-IgG (FIG. 23).

Example 17 Inhibition of VEGF-KDR Internalization by Binding of 6A6Antibody to KDR on Cell Surface

HUVEC cells were placed on a gelatin-coated cover slip at aconcentration of 2×10⁴ cells/well, after 24 hours, the cells were washedtwice with M199 medium and cultured in M199 medium (containing 1% FBS)for 6 hours. The HUVEC cells were allowed to react with variousconcentrations of the antibody for 30 minutes and allowed to react with10 ng/ml of VEGF for 10 minutes. After the completion of the reaction,the cells were immobilized and infiltrated with methanol or 2%formaldehyde for 10 minutes and washed with PBS. Then, the cells wereblocked with 0.1% Triton X-100 and 2% BSA/PBS for 30 minutes, and thecells were allowed to react with a mouse KDR antibody for 1 hour, andthen allowed to react with an FITC-labeled anti-mouse antibody at roomtemperature for 45 minutes. The cover slip was mounted with SloFade(Molecular Probe) and observed with a confocal microscope (Zeiss,Germany) at 488 nm (excitation wavelength).

As a result, as shown in FIG. 24, it was observed that the 6A6 antibodyinhibited the infiltration of KDR into cells, and 6G1 did notsubstantially inhibit the infiltration of KDR into cells.

Example 18 Ex Vivo Analysis of Inhibitory Effect of Anti-KDR-IgG onAngiogenesis

In order to examine whether 6A6-IgG inhibits aortic ring vesselsprouting induced by VEGF, an aortic ring assay was performed. First,arteries were separated from 6-week-old rats (Sprague Dawley), and thencut to a size of about 0.5 mm. The cut artery was placed on 120 μl of amatrigel-coated, 48-well plate and covered with 50 μA of matrigel. VEGF(10 ng/ml) and each of 6A6-IgG, 6C1-IgG and 1121-IgG were mixed withhuman endothelial serum-free medium (Invitrogen) to a final volume of200 μl, and the mixture was placed in each well of the plate. After 6days, the cells were immobilized and stained with Diff-Quick (BaxterDiagnostics). Data were rated on a scale of 0 (least positive) to 5(most positive), and six independent tests were performed. FIG. 25Ashows a vessel sprouting image, and FIG. 25B shows the statisticalresults of scores for vessel sprouting. The 6A6 antibody inhibitedvessel sprouting induced by VEGF, but the 6C1 antibody did not show theinhibitory ability. The above-described ex vivo rat aortic ring assayresults have a very important meaning in addition to the simple factthat 6A6 inhibits angiogenesis induced by VEGF. That is, the resultsrevealed that 6A6 could bind to Flk-1 to neutralize the human KDRhomologue Flk-1 expressed in rats, although it was prepared for thepurpose of neutralizing human KDR. In other words, it can be seen thatthe 6A6 antibody has cross-reactivity between humans and rats.

Example 19 Effect of 6A6 on Inhibition of In Vivo Angiogenesis Inducedby VEGF (in Vivo Mouse Matrigel Plug Assay)

Following the rat aortic ring assay, a mouse matrigel plug assay wasperformed in order to examine whether the human KDR neutralizingantibody 6A6 can inhibit angiogenesis induced by VEGF in vivo in mice.For this purpose, 6-8-week-old C57/BL6 mice were subcutaneously injectedwith 0.6 ml of matrigel, containing 200 μg of the antibody, 100 ng ofVEGF and 10 units of heparin. After 7 days, the matrigel plug was takenout by surgery, and the image thereof was photographed (FIG. 26A). Then,the plug was rapidly frozen with liquid nitrogen in the presence of anOCT (optimum cutting temperature) compound and cut to a thickness of8-12 μm. The cut plug was fixed with 4% neutral bufferedparaformaldehyde, and the density of the microvessels was measured withan anti-CD31 antibody (FIG. 26B). It was observed that the 6A6 antibodycould inhibit blood vessel formation induced by VEGF in vivo in mice.Like the rat ex vivo experiment, it was confirmed again that the 6A6antibody could neutralize the mouse KDR homologue Flk-1 and hadcross-reactivity between humans and mice. Among therapeutic antibodies,a KDR antibody having cross-reactivity between humans and mice has notyet been reported. When the species cross-reactivity of the 6A6 antibodyis used, the in vivo effect of the antibody can be confirmed using miceor rats.

Example 20 Anti-Cancer Effect of 6A6 Antibody in Colon Cancer XenograftAnimal Model

The anticancer effect of the 6A6 antibody in colon cancer xenograftanimal models was analyzed using T cell-, B cell- and NK cell-deletedNOD/SCID IL-2R null mice (female, 11-week-old, weighed 25 g, The JacksonLaboratories, USA) known to have an advantage that the cells more easilyreceive human cancer cells, compared to NOD/SCID mice.

As human cancer cells, human colon cancer cells known as HCT116 (ATCC,USA) were used, and the injection of the tumor cells was performed byinjecting the cells subcutaneously into the left side of the mice at aconcentration of 2×10⁵ cells (serum-free DMEM)/10 μl at day 0.

The 6A6 antibody was injected intravenously into the mice from day 1 (24hours from day 0 at which the tumor cells were injected) three times aweek. The mice were divided into three groups, each consisting of 5animals. The group 1 was a PBS-injected group (control group), the group2 was injected with 100 μg/ea (=4 mg/kg) of the 6A6 antibody, and thegroup 3 was injected with 200 μg/ea (=8 mg/kg) of the 6A6 antibody. Thesize of a tumor occurring in the mice was measured according to thefollowing equation on alternate days for 26 days:Tumor volume=1/2×(length×area×height).

At day 30, the mice were sacrificed, and the weight of a tumor wasmeasured. As a result, it was observed that the tumor size wasdose-dependently reduced in the groups administered with the 6A6antibody, compared to the control group injected with PBS (FIG. 27 andFIG. 28).

Example 21 Anti-Cancer Effect of 6A6 Antibody in Lung Cancer XenograftAnimal Models

Human lung cancer A549 cells (ATCC, USA) were injected subcutaneouslyinto nude mice (Japan SLC, Japan) at a concentration of 7×10⁷ cells toform tumors. 10 days after the injection of the cancer cells, the tumorscould be visually observed, and then the 6A6 antibody was injectedintraperitoneally into the mice three times a week.

The mice were divided into three groups, each consisting of fiveanimals. The group 1 was a PBS-injected group (control group), the group2 was injected with 1 mg/kg of the 6A6 antibody, and the group 3 wasinjected with 1 mg/kg of Avastin (Genentech, USA). As a result, as canbe seen in FIG. 29, the growth of the tumor was inhibited in the groupinjected with the 6A6 antibody and the positive control group injectedwith Avastin, compared to the control group injected with PBS.

Example 22 In Vivo Tumor Targeting of Radioactive Iodine-Labeled 6A6Antibody

The tumor targeting of the 6A6 antibody was analyzed using the bindingaffinity of the antibody to CML (chronic myelogenous leukemia) K562cells. The antibody was labeled with radioactive iodine-125 using aniodobead method to label more than 90% of the antibody with iodine (FIG.30A), and an anti-KDR (6A6) antibody labeled with radioactive iodinehaving a purity of more than 98% was prepared (FIG. 30B). A CML tumormodel was prepared by injecting K562 cells subcutaneously into Balb/cnude mice, and when the tumor size reached 1 cm at 21-28 days after theinjection of the K562 cells, the iodine-125-labeled antibody (100 μg)was injected into the tail vein of the K562 tumor model nude mice. 2hour and 24 hours after the injection of the antibody, gamma-cameraimages of animals having tumors formed therein were obtained. It wasobserved that the introduction of the antibody into the tumors showedsimilar patterns at 2 hours and 24 hours, and the backgroundradioactivity was reduced after 24 hours. It was observed that theantibody was localized to the tumor, suggesting that KDR was expressedon the K562 tumor. Thus, due to the therapeutic effect of the antibodyitself by localization, as well as due to an increase in the therapeuticeffect caused by beta-rays emitted from the radioactive isotope, theantibody can possibly be used as a radioimmunotherapy agent (FIG. 31).

Example 23 Affinity Maturation of 6A6-IgG Using Light Chain Shuffling

In order to identify antibodies having an affinity higher than that of6A6, a heavy chain was removed from the DNA of the complete humanantibody library, prepared in Example 2, using restriction enzyme SfiI.Into the site from which the heavy chain has been removed, a heavy chainof pAK-6A6 treated with SfiI restriction enzyme was inserted. Theresulting DNA was transformed into ETB (Electro Ten blue) cells(Stratagene, USA), and the cells were cultured in SOB medium for 1 hour.Then, the cells were spread on a 2×YT (Cm) square plate, and the nextday, the colony was collected and stored at −70° C. As a result, a 6A6light chain shuffling library having a diversity of 4×10⁶ wasconstructed (FIG. 32).

In order to examine whether the light chain shuffling of the library hasbeen successfully achieved, 48 clones were randomly selected, and thelight chain sequences thereof were analyzed. As a result, there was nooverlap in the light chain sequences of the 48 clones.

From the library, clones having a binding affinity higher than that of6A6 were screened in the same manner as in Example 3 through abiopanning process using a phage display.

18 candidates were finally obtained through the following procedures.

(1) In order to prevent 6A6 from being selected again during thebiopanning process, the DNA of the 6A6 light chain shuffling library wastreated with a restriction enzyme SpeI having a recognition site at theCDR3 of 6A6. After the DNA was transformed into ETB cells, a sub-librarywas constructed based on the cultured cells, and the KDR affinity of thesub-library was analyzed in ELISA. Among candidates resulting from thefourth panning, 94 candidates were selected and subjected to KDR bindingassays in the same manner as in Example 5. Among candidates showingpositive responses, 4 candidates were randomly selected, and the DNAsequences thereof were determined. Also, the candidates were subjectedto VEGF competition assays using the respective phages (FIG. 33). As aresult, 4SD5, 4SC3 and 4SC5, which had KDR neutralizing ability similarto equal to that of the positive control group 6A6, were selected.

(2) In a washing step in the biopanning process, KE3, KE6, 2KG8, 3KE11,3KF11, 3KG3 and K3F1 were selected through competition with soluble KDR.The biopanning process used herein was as follows. Maxisorp Star tubes(Nunc, Denmark) were coated with 4 ml of KDR (5 μg/ml) and blocked with2% skimmed milk/PBS at 37° C. for 2 hours. Then, 500 μl of the 6A6 lightchain shuffling library phage suspended in 2% skimmed milk was allowedto bind to the KDR, and then allowed to react in 0.1% PBS-T (tween20)for 1 hour. Then, the tubes were washed 10 times with 0.1% PBST andwashed 10 times with PBS buffer. Then, 4 ml of soluble KDR (25μg/ml/PBS) was added and allowed to bind thereto for 30 minutes, and thetubes were treated with 100 mM triethylamine for 10 minutes to elute thephage. The eluted phage was neutralized with 500 μl of 1M Tris-Cl(pH7.5) and transformed into E. coli XL1-Blue cells for 50 minutes, andthen the cells were cultured.

The antibodies selected in each of the three panning steps weresubjected to VEGF competition assays in a ScFv-phage particle state.Among four candidate antibodies obtained through the first panning, KE3and KE6, having high VEGF competitive power compared to those of theother candidate antibodies, were selected, and KC7 and KQ11 wereexcluded (FIG. 34A). Among three candidate antibodies obtained throughthe second panning, 2KE5 having low VEGF competitive power was excluded,and the remaining 2KG4 and 2KG8, having VEGF competitive power similarto that of 6A6, were selected. The 2KG4 showed the same sequence as3KG3, an antibody selected later, and thus it was substituted with 3KG3(FIG. 34B).

Among five candidate antibodies obtained through the third panning, 3KG2and 3KF7, having low competitive power, were excluded, and only 3KE11,3KF11 and 3KG3 were selected (FIG. 34C). Similarly, K3F1 obtainedthrough the third panning was selected, and K3F1 showed a significantlyhigh VEGF competitive power compared to that of 6A6 (FIG. 34D).

(3) In a step of allowing phage to bind to the antigen KDR in thebiopanning process, IMC-1121 IgG obtained in Example 8 was also addedand, as a result, IE4, 31G11, 3IG12, 3IE1, 3IH2, I2F2, I3A12 and I3F2clones were selected. The biopanning process used herein was as follows.

Maxisorp Star tubes (Nunc, Denmark) were coated with 4 ml of KDR (5m/ml) and blocked with 2% skimmed milk/PBS at 37° C. for 2 hours. Then,500 μl of the 6A6 light chain shuffling library phage suspended in 2%skimmed milk containing 21 μg/ml of IMC-1121 IgG (0.14 μM) was allowedto bind thereto for 1 hour, and then allowed to react in 0.1% PBST for 1hour. Then, the tubes were washed 10 times with 0.1% PBST and washedwith 10 times with PBS buffer. Then, 4 ml of soluble KDR (25 μg/ml/PBS)was added to the tubes and allowed to bind for 30 minutes, and thentreated with 100 mM triethylamine for 10 minutes to elute the phage. Theeluted phage was neutralized with 500 μl of 1M Tris-Cl (pH 7.5), andthen transformed into E. coli XL1-Blue cells for 50 minutes, and thecells were cultured.

The antibodies selected in each of the three panning steps weresubjected to VEGF competition assays in a ScFv-phage particle state.Among three candidate antibodies obtained through the first panning,only IE4 having VEGF competitive power similar to that of 6A6 wasselected (FIG. 35A). IE4 was analyzed for the DNA sequence thereof and,as a result, 28 amino acids in IE4 were different from those in 6A6. Inaddition, 6A6 had 108 light chain amino acids, whereas IE4 had 107 aminoacids, indicating that one amino acid was deleted in the CDR3 of 6A6.

FIG. 35B shows the results of VEGF competition assays of three candidateantibodies obtained through the third panning. In DNA sequencing, 31G8had a light chain sequence completely different from that of 6A6, and inthe results of FACS, 31G8 did not bind to living cells, indicating thatit was not converted in the form of IgG. 3IG11, 3IG12, 3IE1 and 3IH2were selected, and 3IA7 was excluded, because it had a stop codon in thelight chain sequence. FIG. 35C shows the results of VEGF competitionassays of candidate antibodies obtained through the second panning andthe third panning. 3IA12, I3F2 and I2F2 were all selected.

The light chain DNA sequences of the 18 selected clones are shown in SEQID NO: 164 to SEQ ID NO: 181, and amino acid sequences deduced from theDNA sequences are shown in SEQ ID NO: 2 to SEQ ID NO: 19. Regionssubstituted compared to the light chain amino acid of 6A6 (TTAC-0001)are shown in Table 7. Also, the clones were renamed “TTAC-0002 toTTAC-0019” (Table 8).

TABLE 7 Mutation site of 18 selected clones Clone name Mutation site KE3S13A, R23G, L27I, D29S, V30Q, N31S (TTAC-0002) KE6 S13A, R19G, R23G,N26D, L27I, D29S, V30K, (TTAC-0003) N31S, R38K, M47I, A51S IE4 N1S, F2Y,M3E, V12S, S13A, R19T, R23E, (TTAC-0004) D25K, L27I, D29S, V30K, N31S,R38K, V46L, M47I, A51Q, G56A, G67D, T69M, G76R, E78A, D91G, R92N, T93G,S94K, E95V, T99G, V103L SD5 S13A, T100A (TTAC-0005) 2KG8 S13A(TTAC-0006) 3KE11 T71I (TTAC-0007) 3KF11 P8S (TTAC-0008) 3KG3 P8H(TTAC-0009) 3IG11 V46I (TTAC-0010) 3IG12 R23M (TTAC-0011) 3IE1 P8S,S13P, P39R, Y96F (TTAC-0012) 3IH2 V12L, K16Q (TTAC-0013) K3F1 S13A, K16Q(TTAC-0014) I2F2 S9A (TTAC-0015) I3A12 E59K (TTAC-0016) I3F2 M47I(TTAC-0017) 4SC3 S94N (TTAC-0018) 4SC5 N1Q, M3V, S13A, R23G, D25N, L27I,D29S, (TTAC-0019) V30K, N31S, R38K, M47I, A51S, S66F, G76R, R92S, T93S,S94R, E95D

TABLE 8 New name of antibodies developed Name of antibodies developedNew name 6A6 TTAC-0001 KE3 TTAC-0002 KE6 TTAC-0003 IE4 TTAC-0004 SD5TTAC-0005 2KG8 TTAC-0006 3KE11 TTAC-0007 3KF11 TTAC-0008 3KG3 TTAC-00093IG11 TTAC-0010 3IG12 TTAC-0011 3IE1 TTAC-0012 3IH2 TTAC-0013 K3F1TTAC-0014 I2F2 TTAC-0015 I3A12 TTAC-0016 I3F2 TTAC-0017 4SC3 TTAC-00184SC5 TTAC-0019

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides a fullyhuman antibody, which has an excellent ability to neutralize VEGFreceptor in cells and in vivo, and a composition for inhibitingangiogenesis and a composition for treating cancer, which contain saidantibody. The inventive 6A6 antibody neutralizing vascular endothelialgrowth factor receptor shows excellent neutralizing ability in livingcells, compared to that of a commercially available antibody againstvascular endothelial growth factor receptor, and shows the ability toneutralize vascular endothelial growth factor receptor not only inhumans, but also in mice and rats. Thus, the 6A6 antibody will be usefulin anticancer studies and will be highly effective in cancer treatment.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

The invention claimed is:
 1. A single chain variable fragment (ScFv)molecule comprising: a light chain variable region comprising the aminoacid sequence of any one of SEQ ID NOS: 12 and 16 to 19, and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:20 wherein the ScFv molecule functions to neutralize vascularendothelial growth factor receptor.
 2. A composition for inhibitingangiogenesis comprising: the ScFv molecule of claim
 1. 3. A compositionfor treating cancer comprising: the ScFv molecule of claim
 1. 4. An IgGantibody comprising: a light chain variable region comprising the anamino acid sequence of any one of SEQ ID NOS: 12 and 16 to 19, and aheavy chain variable region comprising the an amino acid sequence of SEQID NO: 20 wherein the antibody functions to neutralize vascularendothelial growth factor receptor.
 5. A composition for inhibitingangiogenesis comprising the IgG antibody of claim
 4. 6. A compositionfor treating cancer comprising the IgG antibody of claim 4.